EP3194623B1 - Systeme und verfahren zur erkennung von stress von stammzellen und verwendungen davon - Google Patents

Systeme und verfahren zur erkennung von stress von stammzellen und verwendungen davon Download PDF

Info

Publication number
EP3194623B1
EP3194623B1 EP15831338.7A EP15831338A EP3194623B1 EP 3194623 B1 EP3194623 B1 EP 3194623B1 EP 15831338 A EP15831338 A EP 15831338A EP 3194623 B1 EP3194623 B1 EP 3194623B1
Authority
EP
European Patent Office
Prior art keywords
stress
differentiation
rex1
expression
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP15831338.7A
Other languages
English (en)
French (fr)
Other versions
EP3194623A4 (de
EP3194623A1 (de
Inventor
Elizabeth PUSCHECK
Daniel A. RAPPOLEE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wayne State University
Original Assignee
Wayne State University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wayne State University filed Critical Wayne State University
Publication of EP3194623A1 publication Critical patent/EP3194623A1/de
Publication of EP3194623A4 publication Critical patent/EP3194623A4/de
Application granted granted Critical
Publication of EP3194623B1 publication Critical patent/EP3194623B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure provides systems and methods (S/M) to detect stress in stem cells.
  • S/M of the invention including assays and high throughput screens, can be used to identify compounds or other potential stressors that can negatively affect development potential.
  • Stem cells are unspecialized cells that are able to renew themselves through cell division for long periods. Stem cells can be subdivided and classified on the basis of their potency.
  • a totipotent stem cell is produced from fusion between an egg and a sperm. Stem cells produced by the 1 st few divisions of the fertilized egg cell are also totipotent stem cells. These cells can grow into any type of cell that makes up the developing organism, extra-embryonic tissues (e.g., yolk sac and placenta) and embryo.
  • Pluripotent stem cells are descendants of totipotent cells that can differentiate into any cell type except for placental cells.
  • Multipotent stem cells are descendants of pluripotent stem cells that can produce only cells of a closely related family of cells (e.g.
  • blood cells such as red blood cells, white blood cells and platelets, or all placental lineages).
  • Progenitor (sometimes called unipotent) cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells.
  • the 1 st approach is to differentiate stem cells by removing growth factors that support potency to test stress effects during production of differentiated, functional cell lineages.
  • a drawback of this approach is that single lineage tests may find toxicants for only that particular lineage.
  • the second approach is to measure outright lethality and morbidity of stem cells in the presence of potency-maintaining growth factors. This approach, however, does not identify compounds or events that stress, but do not kill stem cells. That is, this approach does not identify lower, submorbid stress doses that lead to maladaptive runting and toxic responses to the embryo that do not necessarily kill stem cells.
  • Puschek et al., Fertil. Steril. (2013) 100 (Supplement): S456 discloses the cultivation of murine trophoblast stem cells (TSCs) in the presence of FGF-4. After the reduction of oxygen in the presence of FGF-4, the stress response is measured by determining the activities of AMPK and SAPK.
  • the invention is defined in the claims. Any subject-matter which is described herein, but which is not claimed, does not form part of the invention.
  • the current disclosure provides S/M to detect stress in stem cells where the detected stressor might affect all stem cell lineages and might not kill the stem cells. Testing for stress before differentiation occurs is of great benefit because stress at this stage can affect all later-developing cell lineages. Thus, the S/M provide a unifying test.
  • the S/M also can detect stressors that act at sub-morbid runting doses that slow growth and negatively impact and imbalance differentiation of stem cells.
  • the S/M provide for more sensitive stress detection that does not require cell death to observe stress. Imbalances of differentiation forced on cultured stem cells measure organismal responses not needed for adaptation to culture. Thus stress induced differentiation should reduce or replace the need for slower and more costly gestational animal in vivo reproductive toxicology tests.
  • the S/M are based on the finding that stressed stem cells (i) slow their growth and (ii) differentiate under physiological conditions that would normally maintain their toti- or pluri-potency. That is, they differentiate earlier than they normally would. Early differentiation of stem cells depletes the number of stem cells available to differentiate later in the development process, reducing the number of stem cells available to form organ systems that arise later during development. This type of early, stress-induced differentiation is referred to as "prioritized differentiation.” Prioritized differentiation negatively affects development potential.
  • the disclosure provides numerous S/M to detect stress in stem cells by measuring potency factors (and markers) and prioritized differentiation factors (and markers).
  • “Potency factors” can be thought of as “brake pedals” for differentiation. That is, their expression and activity is associated with toti- or pluri-potency.
  • “Differentiation factors” can be thought of as “accelerator pedals” for differentiation. That is, their expression and activity is associated with normal differentiation that occurs when potency-maintaining extracellular growth factors are removed. Under stress conditions, however, potency factors can decrease and differentiation factors can increase even in the presence of potency-maintaining extracellular growth factors. This type of differentiation is referred to as “compensatory” or “prioritized” differentiation.
  • stem cells under compensatory or prioritized differentiation, can be induced by stress from a toti- or pluripotent state into a more differentiated/committed cell type despite the presence of growth factors that maintain potency. Stress also slows stem cell growth and induces differentiation in fewer-than-normal stem cells to further deplete stemness to "compensate" for fewer cells. Compensatory or prioritized differentiation of stem cells despite potency-maintaining growth factors also prioritizes differentiation to emphasize creating sufficient 1 st lineage differentiation at the sacrifice of later lineages.
  • nuclear differentiation factors Decrease in nuclear potency factors and an increase in 1 st lineage determining nuclear differentiation factors provide (early lineage>later lineage) "markers" that are evidence that differentiation is enabled, but their activity does not necessarily lead to or cause the potent or differentiated state.
  • the differentiated state requires sufficient loss of nuclear potency factors and/or gain of nuclear differentiation nuclear factors. But, different stressors may more strongly release the brake pedal (decrease potency) or engage the accelerator pedal.
  • Potency factors and/or markers include: Oct4; Sox2; Nanog; Rex1; TEAD4 (TEA domain family member 4); Err ⁇ (estrogen receptor-related- ⁇ ); Cdx2 (caudal homeobox domain); Id2; Klf5 (Krueppel-like factor 5); PDCD2 (programmed cell death 2); CARM1(coactivator-associated-arginine methyltransferase); Sall4 (Spalt-like transcription factor); SSEA1; SSEA3; SSEA4; TRA-1-60; TRA-1-81; and Elf5 (E74-like factor 5 (ets domain transcription factor).
  • Prioritized differentiation factors and/or markers include: Lrp2; Dab2; Fgf5 (fibroblast growth factor 5); Pdgfra (Platelet-derived growth factor receptor A); Sox17 (SRY box containing gene 17); Gata4/6; PAX8 (Paired box 8); NEFL (Neurofilament protein light chain); TSHR (thyroid stimulating hormone receptor); Brachyury; Laminin; AFP (a-feto-protein); Nestin; Goosecoid; MEK1 (mitogen-activated protein kinase kinase 1); MEK2; AKT(RAC-alpha serine/threonine-protein kinase/protein kinase B); PL1 (Placental lactogen-1); Plf (proliferin); PL2 (placental lactogen-2; Hand1; Stra13; Tpbpa; Mash2 (mouse achaete scute 2); Gcm1 (glial
  • the S/M can be used to assess compounds and other environmental conditions for stress effects on stem cells.
  • the S/M can be used to screen compounds and other environmental conditions for stress effects on stem cells before the compounds or other environmental conditions are developed, commercialized or released into the workplace or atmosphere.
  • the S/M of the invention include assays and high throughput screens (HTS).
  • FIGs. 1A-1C SAPK mediates decreased Gcm1 and Tpbpa and increased Hand1 at all O 2 levels. There are greater effects as O 2 deviates from the stemness optimum of 2% O 2 .
  • Trophoblast stem cells TSCs
  • FGF4 was replenished the next day at time zero
  • SAPK inhibitor SP600125 10 ⁇ m or 50 ⁇ M was co-incubated with TSCs for 2 days at the indicated O 2 levels ⁇ SP600125. Markers were quantitated using qPCR.
  • FIG. 1B is a graph derived from data in ( FIG.
  • SAPK mediates suppression of Gcm1 and TPBPa, and induces Hand1 at all O 2 levels, but there are greater effects for these lineage choices at 0.5% O 2 and 20% O 2 and the least effect is seen at 2% O 2 where it has been reported that SAPK activity is lowest.
  • TSCs were grown at 20% O 2 , then passed and switched to 20, 2, or 0.5% O 2 overnight.
  • FGF4 was replenished the next day at time zero, JNKI1 (1 ⁇ M) was preincubated with TSC 3 hr prior to time zero, then cells were cultured for 2 days at the indicated O 2 levels with or without the two SAPK inhibitors.
  • RNA was isolated and quantitated using qPCR. FIG. 3A and FIG. 3B .
  • Nuclear differentiation factors have complex time- and O 2 level-dependent regulation over 7 days of FGF4 removal and TSC differentiation. TSCs were grown at 20%, then passed and switched to 20, 2, 0.5, or 0% O 2 for one day and RNA was isolated to emulate previous studies. FGF4 was removed after one day, then cells were cultured for 1, 2, 4, or 7 days, and RNA was isolated and quantitated using real time TaqMan qPCR.
  • FIG. 3B shows a three dimensional histogram of time in the x axis, O 2 levels in the y axis, and average levels of the four nuclear differentiation factors and one nonnuclear lineage marker in the z axis.
  • FIGs. 4A-4D Multipotency factors undergo rapid loss on day 1 at 0.5% O 2 with FGF4 present, but the least loss after 7 days of FGF4 absence. Terminal differentiation is lowest at 0.5% O 2 and highest at 20% O 2 after 7 days, and labyrinthine placental lineages are dependent on 20% O 2 .
  • FIG. 4A shows averages of loss of a mean of three potency factors
  • FIG. 4B shows the gain of a mean of five differentiation factors during seven days of differentiation caused by removal of FGF4 dependent on four O 2 levels
  • FIG. 4C shows the five differentiation factors averaged in (B), but only at highest differentiation at day 7.
  • FIG. 4D shows the oxygen-dependent accumulation of mRNA of a marker of the 1 st lineage (PL1) and a later lineage (synTA). Note that synTA always requires highest O 2 but PL1 is induced by hypoxic stress early but requires highest O 2 levels at day 7.
  • FIG. 5 Although greatest multipotency factor loss and differentiation factor gain initiate at 0.5% O 2 with FGF4 for 24 h, terminal differentiation is lowest at 0.5% O 2 and highest at 20% O 2 after 7 days when multipotency is lowest. TSCs were grown at 20% O 2 , then passed and switched to 20, 2, or 0.5 O 2 overnight. RNA was isolated and five terminal differentiation marker mRNAs were quantitated using real time TaqMan qPCR.
  • FIGs. 6A-6D The filled in triangles below PL1, Plf and SynTa show tendencies of all three to be dependent on high O 2 at 7 days, but early lineage markers PI1 and Plf are induced by hypoxia at day 1, but not SynTa FIGs. 6A-6D .
  • FIG. 6A TSCs that differentiated without FGF4 for 7 days had the highest ATP levels at 0.5% O 2 and the lowest ATP in the rank order 20% ⁇ 2% ⁇ 0% compared with baseline stem cells at 20% O 2 + FGF4.
  • TSCs were cultured in 0-20% O 2 for 0-7 days ⁇
  • FIGs. 7A-7D Correspondingly, at 7 days, mitochondrial ⁇ ⁇ m increased only at O 2 ⁇ 2% with FGF4 removal. TSCs were cultured for 7 days at 20% O 2 (a,d), 2% O 2 (b,e), or 0.5% O 2 (c,f) with FGF4 (a-c) or without FGF4 (d-f) and stained with JC1. Micron bar in micrograph (a) indicates 50 ⁇ M. Green fluorescence indicates monomeric stain and low membrane charge and red fluorescence indicates polymeric stain and high charge. For original color fluorescence figures see ( Xie, Stem Cell Research, 13:478, 2014 (Xie, 2014)). FIGs. 7A-7D .
  • FGF4 maintains inactive mitochondria at 20%, 2%, or 0.5% O 2 ( FIG. 7A ) and mitochondria display higher mitochondrial membrane potential levels ( ⁇ m ) at 20% > 2% but remain depolarized at 0.5% O 2 after FGF4 removal.
  • TSCs were cultured for 2 days at 20% ( FIG. 7A, FIG. 7C ) or 2 % O 2 ( FIG. 7B, FIG. 7D ) with FGF4 ( FIG. 7A, FIG. 7B ) or without FGF4 ( FIG. 7C, FIG. 7D ) and then stained for ⁇ m using the MitoTracker dye (A). Micron bars in micrograph A indicate 50 ⁇ M.
  • FIGs. 8A-8F are examples of the MitoTracker dye
  • TSCs were cultured for 2 days at 2% O 2 ( FIG. 8A, FIG. 8C, FIG. 8E ) or 20 % O 2 ( FIG. 8B, FIG. 8D, FIG. 8F ) with FGF4 ( FIG. 8A, FIG. 8B ) or without FGF4 ( FIG. 8C-FIG. 8F ) and then stained with the probe JC1 or TMRM as indicated, in the presence or the absence of uncoupler FCCP as a control. Micron bars in micrograph A indicate 50 ⁇ M. FIGs. 9A-9E .
  • FGF4 maintained COX subunit I and PKM2 in the phosphorylated state at all O 2 levels, but total COX and PKM2 levels are regulated by hypoxia and did not change with FGF4 removal.
  • TSCs were cultured for 2 days ⁇ FGF4 at 0-20% O 2 and assayed by immunoblot for phosphorylated cytochrome c oxidase (pCOX) using a phospho-specific antibody ( FIG. 9A ), total COX ( FIG. 9B ), actin b (ACTB) and, pPKM2 ( FIG. 9C ), PKM1 ( FIG. 9D ), or PKM2 ( FIG. 9E ).
  • pCOX phosphorylated cytochrome c oxidase
  • FIGs. 10A and 10B Histograms show the pCOX, total COX levels, PKM1, PKM2, and pPKM2 normalized to ACTB, with bars showing the average of 3 experiments ⁇ s.e.m.
  • FIGs. 10A and 10B In TSCs cultured for 7 days at 20% O 2 , all terminal differentiated lineages were O 2 sensitive, but PL2 and CtsQ were most inhibited by the mitochondrial inhibitors cyanide and antimycin A, and PL2 was most highly induced by the mitochondrial activator DCA at 2% O 2 .
  • FIG. 10A TSCs were incubated for 7 days ⁇ FGF4 at 20%, 2%, or 0.5% O 2 with the mitochondrial activator DCA.
  • TSCs were cultured for 7 days at 20% O 2 , ⁇ FGF4, and with mitochondrial inhibitors antimycin A or cyanide ( FIG. 10B ).
  • the cells were then lysed and assayed by qPCR for the relative levels of 5 terminal differentiation markers PL1, PL2, Plf, CtsQ, and synTa.
  • the experiment was repeated 3 times and bars show the average ⁇ s.e.m., (a) indicates no significant difference and (b) indicates a significant difference (p ⁇ 0.05) between TSCs with FGF4 removed without or with mitochondrial agonist or antagonist. Where changes in markers are significant (b), the increase or decrease due to mitochondrial agonist or antagonist is shown above the histogram bar.
  • FIG. 11 shows the increase or decrease due to mitochondrial agonist or antagonist above the histogram bar.
  • FIG. 12 TSCs produce twice as much ROS (H 2 O 2 ) 48 hr after FGF4 removal.
  • FIGs. 13A-13F Hyperosmotic stress effects on cell proliferation in murine embryonic stem cells (mESCs).
  • mESCs murine embryonic stem cells
  • FIG. 13B mESCs were cultured ⁇ leukemia inhibitory factor (LIF) and ⁇ 200 mM sorbitol for 0 - 72 hr. mESCs were trypsinized and counted with a hemocytometer following trypan blue exclusion. Error flags represent standard error of the mean, n ⁇ 3.
  • FIG. 13C Doubling rates were calculated and tabulated from cell counts in ( FIG. 13B ).
  • FIG. 13D mESCs were incubated in the presence of LIF ⁇ 200 mM sorbitol for 24, 48, or 72 hr and transmitted light micrographs were taken.
  • mESCs were cultured in the presence of 200 mM sorbitol for 0-24 hr.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • n 3
  • Time-matched cells were fixed, and stained for the cleavage product of caspase 3 with Hoechst staining of the nuclei.
  • FIG. 14 Activation of p38, MEK1/2, c-Jun N-terminal kinase (JNK), and AKT by hyperosmolarity. mESCs were incubated in 200 mM sorbitol for 0-60 min to detect kinetics of enzyme activation.
  • mESCs were incubated for 4 hr in the presence of 200 mM sorbitol with or without one of the enzyme inhibitors SB202190 (p38), PD98059 (MEK1), U0126 (MEK1/2), LJNKi-1 (JNK), AKTi (AKT), or LY294002 (PI3K).
  • Cells were lysed and proteins were fractionated using SDS-PAGE, blotted, and probed for phospho-p38 (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho-ERK1/2 (Thr202/Tyr204), or phospho-AKT (Ser473). Blots are representative of triplicate experiments.
  • FIGs. 16A-16E are representative of triplicate experiments.
  • FIG. 16A The inner cell mass (ICM) of the blastocyst coexpresses Oct4/Rex1/Nanog in pluripotent cells at E3.5. After E3.5 a Rex1 subpopulation delaminates from the ICM and expresses primitive endoderm (PrEndo) marker, Lrp2, at E3.5 and extraembryonic endoderm (ExEndo) marker, Dab2, at E4.5. At E4.75, Rex1 decreases in the remaining cells of the ICM and, by E5.5, fibroblast growth factor (FGF)5 is expressed in embryonic ectoderm (EmEcto).
  • FGF fibroblast growth factor
  • FIG. 16B Upward arrow indicates that expression is maintained or increasing; downward arrow means that expression is decreasing or absent.
  • FIG. 16B mESCs were incubated in 200 mM sorbitol for 0-24 hr and lysed. Proteins were fractionated using SDS-PAGE, blotted, and probed for Oct4, Sox2, Nanog, or Rex1. Histograms show relative expression of each protein when normalized to amido black.
  • FIG. 16C Total RNA was isolated and subjected to reverse transcription to form complementary DNA (cDNA). Real-time quantitative PCR (RTqPCR) was performed with Oct4, Nanog, and Rex1 primers. Histograms represent relative fold change in mRNA expression using the ddCt method.
  • FIG. 16C Total RNA was isolated and subjected to reverse transcription to form complementary DNA (cDNA). Real-time quantitative PCR (RTqPCR) was performed with Oct4, Nanog, and Rex1 primers. Histograms represent relative fold change in mRNA expression using
  • FIG. 17 MG132 proteasome inhibitor effects on Oct4, Nanog, and Rex1 during sorbitol stimulation of mESCs. mESCs were treated for 4 hr with 0 or 200 mM sorbitol ⁇ 10 ⁇ M MG132 and then lysed. Total cellular protein was fractionated using SDS-PAGE, blotted, and probed for Oct4, Nanog, or Rex1.
  • FIGs. 18A-18D Lactacystin effects on Oct4, Nanog, Rex1 during sorbitol stimulation of mESCs. mESCs were treated for 4 hr ⁇ 200 mM sorbitol ⁇ 25, 50 ⁇ M lactacystin, then photographed before lysing. FIG. 18A, FIG.
  • Enzymes activated during 4 hr of hyperosmotic stress initiate the differentiation program in mESCs.
  • mESCs were incubated in 200 mM sorbitol for 0-4 hr with or without the presence of ( FIG. 19A ) PD98059 (10 ⁇ M), or ( FIG. 19B ) LY294002 (25 ⁇ M), L-JNKi-1 (2 ⁇ M), or U0126 (40 ⁇ M).
  • mESCs were lysed, and proteins were fractionated using SDS-PAGE, blotted, and probed for Oct4, Nanog, or Rex1. Histograms show relative expression of each protein when normalized to amido black expression. Error flags represent standard error of the mean (n ⁇ 3).
  • FIGs. 20A-20C p38MAPK rescues pluripotency of mESCs during 24 hr of hyperosmotic stress.
  • FIG. 20A mESCs were incubated ⁇ 200 mM sorbitol for 0-24 hr and ⁇ p38 inhibitor SB202190 (10 ⁇ M). mESCs were lysed, and proteins were fractionated using SDS-PAGE, blotted, and probed for Oct4 or Nanog.
  • FIG. 20A mESCs were incubated ⁇ 200 mM sorbitol for 0-24 hr and ⁇ p38 inhibitor SB202190 (10 ⁇ M). mESCs were lysed, and proteins were fractionated using SDS-PAGE, blotted, and probed for Oct4 or Nanog.
  • FIG. 20C mESCs were incubated ⁇ 200 mM sorbitol for 0-24 hr ⁇ either U0126 or LY294002 (25 ⁇ M). mESCs were lysed and proteins were fractionated using SDS-PAGE, blotted, and probed for Oct4 or Nanog. Histograms show relative expression of each protein when normalized to amido black expression. Error flags represent standard error of the mean (n ⁇ 3).
  • FIGs. 21A and 21B JNK-dependent signaling suppressed Rex1 expression during 24 hr of hyperosmotic stress.
  • FIG. 21A mESCs were incubated ⁇ 200 mM sorbitol for 0-24 hr ⁇
  • A p38MAPK inhibitor SB202190 (10 ⁇ M) or PI3K inhibitor LY294002 (25 ⁇ M); or JNK inhibitor L-JNKi-1 (2 ⁇ M).
  • mESCs were lysed, and proteins were fractionated using SDS-PAGE, blotted, and probed for Rex1. Histograms show relative expression of each protein when normalized to amido black expression. Error flags represent standard error of the mean (n ⁇ 3). ' '*' ' denotes significant difference from unstressed mESCs. ' 'a' ' indicates significant difference from stress-only time point. ANOVA and Student-Newman-Keuls post hoc tests (P ⁇ 0.05). ( FIG.
  • FIGs. 22A-22L Viable pluripotency activity reporters respond to stress treatment as previously reported for endogenous pluripotency reporter protein levels. ESCs were lentivirus-infected (Step 1; FIG. 22A, FIG. 22B, FIG. 22C ), FACS-selected (Step 2; FIG. 22D , FIG. 22E, FIG.
  • FIG. 22F stress-tested (Step 3) without ( FIG. 22G, FIG. 22H , FIG. 22I ) or with 200mM sorbitol for three days ( FIG. 22J, FIG. 22K, FIG. 22L ) using pluripotency reporter Oct4-GFP ESCs ( FIG. 22A, FIG. 22D , FIG. 22G , FIG. 22J ), Rex1-RFP ESCs ( FIG. 22B , FIG. 22E , FIG. 22H , FIG. 22K ), or ESCs with both reporters ( FIG. 22C , FIG. 22F , FIG. 22I, FIG. 22L ).
  • FIG. 22A, FIG. 22D , and FIG. 22G indicate 100 ⁇ m as FIG. 22A-C
  • FIG. 22G-I and FIG. 22J-L were micrographed at 10X
  • FIG. 22D-F were micrographed at 20X.
  • FIGs. 23A and 23B Stress induces loss of Rex1-RFP but not Oct4-GFP after days of 200mM sorbitol.
  • Viable pluripotency reporter Rex1-RFP ESCs ( FIG. 23A ) or Oct4-GFP ESCs ( FIG. 23B ) were cultured in 94-well plates to 30% confluence.
  • FIG. 24 Viable Rex1 promoter reporter responds with stress treatment in a dose-dependent manner in flow cytometry assay.
  • Rex1 promoter RFP reporter ESCs respond to stress treatment with a dose-dependent 2.8-fold increase in the low Rex1 activity subpopulation and a 20% decrease in high activity Rex1 subpopulation.
  • Rex1-RFP mESCs expressing Rex1 promoter reporter were cultured in 12-well plates until 30% confluent, treated with OmM, 200mM, and 300mM sorbitol for 3 days, trypsinized, and cell suspensions were assayed by flow cytometry.
  • Arbitrary thresholds vertical red lines
  • FIG. 25A Areas in parental dim, intermediate dim, and bright were defined by the 2 vertical red lines and were quantified as pixels using Image J from data in ( FIG. 25A ) to give histograms in ( FIG. 25B ).
  • the histogram bars ( FIG. 25B ) are derived from three independent experiments (see also FIG. 26A and 26B ) with errors bars showing s.e.m, One-way ANOVA was performed followed by Dunnett's post hoc test; (a) indicates significantly smaller bright cell subpopulation and greater parental dim cells at 300mM sorbitol (a) (P ⁇ 0.05), but no significant decrease in bright cell subpopulation or increase in parental dim cells at 200mM sorbitol (b).significant.
  • FIGs. 26A and 26B are derived from three independent experiments (see also FIG. 26A and 26B ) with errors bars showing s.e.m, One-way ANOVA was performed followed by Dunnett's post hoc test; (a) indicates significantly smaller bright cell subpopulation and greater parental dim
  • Viable Rex1 promoter reporter responds with stress treatment in a dose-dependent manner in flow cytometry assay.
  • mESCs expressing Rex1 promoter reporter were cultured in 12-well plates till 30% confluence. Cells were then treated with 200-300 mM of sorbitol in 25 mM increments for 3 days. After trypsinization, the cell suspensions were subjected for flow cytometry assay. An arbitrary threshold was drawn at 5x10 3 on the x-axis and another near the low point between the bright peak and the dimmer peak to its left ( FIG. 26A ). This created three areas of non-transgenic Parental ESC dim at the level of ESCs that do not express Rex1-RFP (bottom graph of the four).
  • FIG. 26B The histogram bars ( FIG. 26B ) are derived by three independent experiments, one of which is shown in ( FIG. 26A ), errors bars showing s.e.m, One-way ANOVA was performed followed by Dunnett's post hoc test using OmM as reference for parental dim, intermediate dim, and bright separately. (a) indicates significant decrease in bright subpopulations and increase in parental dim subpopulations at 250-300mM sorbitol (P ⁇ 0.01) and (b) indicates no significance at 200-225mM sorbitol for changes in Rex1-RFP bright or parental dim subpopulations.
  • FIG. 27 The histogram bars ( FIG. 26B ) are derived by three independent experiments, one of which is shown in ( FIG. 26A ), errors bars showing s.e.m, One-way ANOVA was performed followed by Dunnett's post hoc test using OmM as reference for parental dim, intermediate dim, and bright separately. (a) indicates significant decrease in bright subpopulations and increase in parental dim subpopulation
  • Rex1 promoter RFP have a stress dose-dependent loss of endogenous Rex1 protein corresponding to dose-dependent loss of Rex1 activity shown in FIG. 24 .
  • (B) One-way ANOVA was performed followed by Dunnett's post hoc test; (a) indicates significantly lower endogenous Rex1 at 225-300mM sorbitol (P ⁇ 0.05) but not at 200mM sorbitol (b).
  • FIG. 28 Partial validation of the Theory of Compensatory Differentiation whereby stress decreases in size the normal levels of stem cell expansion and, in the nonmorbid stress range where doubling rates were near-normal, the diminished potency group that is enabled to differentiate is constant in size.
  • Sorbitol (mM) 0 200 225 250 275 300 LIF+ % # % # % # % # % # % # Bright 833 77.1 756 72 71.9 61.8 Intermediate 8.1 10.7 10.7 11.4 11 13.8 Dim 86 12.2 13.7 16.6 17.1 264 24.4 Intermediate+ Dim 16.7 22.9 24.4 28 28.1 28.1 %Dim+intermediate# X Cell# 16.7 x 460 22.9 x 432 24.4 x 407 28 x 356 28.1 x 264 28.2 x 161 7683 9893 9930 9968 7418 4540 Total % / # 100 460 100 432 100 407 100 356 100 264 100 161 Sorbitol Duration Relative Cell # Doubling rate Intermediate and very dim (%very low potency) x cell number OUTSIDE BOX++ INSIDE BOX cell number++ 0 0 100 0 72 460 32.7hr 200 72 432 34,1 hr 9893 225 72
  • the invention is defined in the claims. Any subject-matter which is described herein, but which is not claimed, does not form part of the invention.
  • the present disclosure provides systems and methods (S/M) to detect stress in stem cells and uses thereof.
  • S/M can detect sub-morbid stress in stem cells that can lead to stem cell depletion and predict later organismal toxicity and morbidity.
  • Stem cells are cells that are totipotent, pluripotent, or multipotent and are capable of differentiating into one or more different cell types.
  • Stem cells may be derived from any organism, e.g. any mammalian species, e.g. human, primate, equine, bovine, porcine, canine, feline, etc. In certain embodiments (CE), they are derived from a human or a mouse.
  • a totipotent stem cell can give rise to all of the embryonic and extraembryonic tissues of an organism.
  • An example of a totipotent stem cell is a fertilized oocyte.
  • a pluripotent stem cell can give rise to progeny that can undergo differentiation, under appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from all of the three germinal layers (endoderm, mesoderm, and ectoderm).
  • Pluripotent stem cells contribute to all tissues of a prenatal, postnatal or adult animal.
  • Cell pluripotency is a continuum, ranging from the completely pluripotent cell that can form every cell of the embryo proper, e.g., ESCs and induced pluripotent stem cells (iPSCs), to the incompletely or partially pluripotent cell that can form cells of all three germ layers but may not exhibit germline transmission or ability to generate a whole organism.
  • a standard art-accepted test to establish pluripotency of a cell population is ability to form a teratoma in 8-12 week old SCID mice.
  • Expression of certain combinations of molecular markers are also pluripotent stem cell characteristics.
  • human pluripotent stem cells express markers including: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog.
  • a multipotent stem cell is capable of differentiating into at least two differentiated cell types.
  • stem cells include embryonic stem cells; cord blood stem cells; umbilical cord matrix stem cells; tissue-specific stem cells; and placental TSCs.
  • Progenitor cells are also stem cells.
  • Embryonic stem cells are stem cells derived from the inner cell mass (ICM) of an early stage embryo known as a blastocyst. Human embryos reach the blastocyst stage 4-5 days post fertilization, at 50-150 cells. ESCs include those derived from an embryo of up to 7 days after fertilization. ESCs are pluripotent and have the capability of proliferating indefinitely in culture, under conditions that allow their proliferation without differentiation.
  • Primate ESCs may be obtained by, e.g., the methods in US Pat. Nos. 5,843,780 and 6,200,806 .
  • Primate and human stem cells may also be obtained from commercial sources such as WiCell, Madison, WI.
  • Mouse placental TSCs like mouse ESCs, are derived from a blastocyst arising at E3.5 (3.5 days after fertilization).
  • the growth factor fibroblast growth factor (FGF)4 is synthesized by the ICM ( Rappolee, Development, 120:2259, 1994 ) and this is necessary to maintain the adjacent polar trophectoderm, source of TSCs, in a stem cell state ( Chai, Dev. Biol., 198:105, 1998 (Chai, 1998)).
  • mice TSCs The isolation of mouse TSCs was 1 st reported in 1998 ( Tanaka, Science, 282:2072, 1998 (Tanaka, 1998)) and used FGF4, the FGF4 cofactor heparin, and conditioned medium from mouse embryonic fibroblasts (MEF-CM) to supply additional necessary cofactors. It has been shown that TGFB family members activin and TGFB1 can replace MEF-CM. Erlebacher, Dev. Biol., 275:158, 2004 ).
  • TSCs In culture, removal of FGF4 enables differentiation of TSC to produce several different lineages that are defined by expression of Hand1, Stra13, PL1, Proliferin, Gcm1, SyncytinA, Ctsq, PL2, Tfeb, Tpbpa, Mash2.
  • TSCs lose the potency factors TEAD4, Cdx2, Id2, and ErrB (reviewed in Puscheck, Adv Exp Med Biol, 843:77, 2015 ).
  • Stem cells used within the scope of the present disclosure can be cultured according to any appropriate method known in the art.
  • An appropriate medium for stem cell culture includes 80% Dulbecco's modified Eagle's medium (DMEM; no pyruvate, high glucose formulation, Gibco BRL), with 20% fetal bovine serum (FBS; Hyclone), 0.1 ⁇ M ⁇ -mercaptoethanol (Sigma), and 1% non-essential amino acid stock (Gibco BRL).
  • DMEM Knockout can also be used.
  • the culture can further be supplemented with Knock-out serum replacement (KSP).
  • KSP Knock-out serum replacement
  • Cultures can also include one or more carbon sources.
  • the cultures include L-analyl-L-glutamine.
  • the cultures can include serum or can be serum-free.
  • stem cells can be cultured with an ALK5 inhibitor and one of a MEK or Erk inhibitor, optionally with a GSK3 inhibitor and/or LIF. Mek and GSK3 inhibitors in one media are called 2i (two inhibitor media) and have been shown to aid in maintaining the ground state of ESCs. Marks, Cell, 149:590, 2012 .
  • the culture media include basal media components for cell survival (e.g., vitamins and minerals, optionally in an isotonic condition).
  • stem cells can be cultured in contact with feeder cells.
  • feeder cells include fibroblast cells, e.g., mouse embryonic fibroblast (MEF) cells, or x-ray inactivated CF1 feeder cells. Methods of culturing stem cells on feeder cells are known in the art.
  • stem cells are cultured in the absence of feeder cells.
  • Stem cells e.g., can be attached directly to a solid culture surface (e.g., a culture plate), e.g., via a molecular tether.
  • exemplary molecular tethers include matrigel, an extracellular matrix (ECM), ECM analogs, laminin, fibronectin, or collagen.
  • the disclosure is based in part on the finding that stress stimuli (e.g., toxicological compounds, drugs under development, environmental pollutants) cause stem cells to differentiate despite the presence of growth factors and an environment that is meant to sustain proliferation and potency of stem cells of the early conceptus (both ESCs and TSCs).
  • stress stimuli e.g., toxicological compounds, drugs under development, environmental pollutants
  • the unique exponential growth phase normally occurs for days after fertilization and before implantation into the uterus, and then one to several weeks after implantation (depending on the mammalian species).
  • ESCs and TSCs increase population size under normal conditions, but have diminished population growth under stressed conditions.
  • the strategy of both ESCs and TSCs during stress is to compensate for stress-diminished population size by further depleting stem cell number by differentiating to the 1 st lineage at the expense of later lineages
  • compensatory differentiation is linked to prioritized differentiation when stress induces the differentiation to 1 st lineage and suppresses later lineages.
  • Compensatory and prioritized differentiation can occur at lower levels of stress that do not cause toxicity (cell death) in individual stem cells, but additively depletes stem cells so that later cell lineages (e.g. heart, liver, etc.) have insufficient stem cells for development. Thus later lineages may be depleted even though toxicants tested may not directly affect these later lineages.
  • the described S/M provide assays that are more sensitive and earlier in ontogeny. The S/M can be practiced in vitro, replacing animals needed in toxicological assays.
  • the assays and high throughput screens (HTS) of the invention can be used to identify compounds or other potential stressors that can negatively affect development potential.
  • “Developmental potential” refers to the capacity of a stem cell (e.g., pluripotent cell) to divide and differentiate and/or the capacity of an embryo to grow according to expected developmental milestones when compared to an average non-stressed stem cell and/or healthy embryo.
  • the S/M can also provide early indicators of miscarriage or at-risk pregnancy; indicator of infertility and/or the effect of therapeutics on fertility; indicator of ESC stress and placental insufficiency as the issue for the infertility; early detection and prediction of diseases of placental deficiency; an in vitro toxicology screen for compounds that may have teratogenic potential.
  • potency factors can be thought of as “brake pedals” and differentiation factors can be thought of as “accelerator pedals” for differentiation.
  • Normal differentiation occurs when extracellular potency-maintaining growth factors (e.g. FGF4 for mouse TSCs or LIF for mouse ESCs) are removed. Stress-induced compensatory and prioritized differentiation occurs despite the presence of these extracellular growth factors.
  • intracellular stress enzymes e.g. SAPK/JNK, and AMPK
  • Potency factors and/or markers for ESC include Oct4; Sox2; Nanog; Rex1; Klf5; PDCD2; CARM1; Sall4); SSEA1; SSEA3; SSEA4; TRA-1-60; and TRA-1-81.
  • Potency factors and/or markers for TSC include TEAD4; Err ⁇ ; Cdx2; Id2; and Elf5.
  • Differentiation factors and/or markers for ESC include Lrp2; Dab2; Fgf5; Pdgfra; Sox17; Gata4/6; PAX8; NEFL; TSHR; Brachyury; Laminin; AFP; Nestin; goosecoid; Cubulin; TBP; Vimentin; and Snail.
  • Differentiation factors and/or markers for TSC include PL1; Plf; PL2; Hand1; Stra13; Tpbpa; Mash2; Gcm1; Tfeb; and Ctsq.
  • Factors and markers with enzyme activity include MEK1; MEK2; AKT; PI3K; AMPK; SAPK/JNK; p38MAPK; GSK3; and JAK/STAT. Sometimes these factors and markers can override brake pedals and accelerator pedals, during runting stress. In CE, MEK1; MEK2; AKT; AMPK; SAPK/JNK; GSK3; and JAK/STAT can be classified as differentiation factors or markers.
  • ESCs Oct4, Nanog, and Rex1 are potency factors that decrease with stress and prioritized differentiation.
  • Pdgfra, Sox17, Dab2, Lrp2, and GATA4/6 are prioritized differentiation markers that increase with 1 st lineage. These markers increase with stress-induced differentiation.
  • Brachyury and FGF5 are later lineage prioritized differentiation markers that decrease with stress and prioritized differentiation.
  • AMPK, SAPK and JNK are intracellular stress enzymes that increase with stress and mediate compensatory and prioritized differentiation.
  • the Oct4 gene also known as Oct3 or POU5F1 belongs to the POU (Pit-Oct-Unc) transcription factor family.
  • the Oct4 protein (octamer-binding transcription factor 4) can activate the expression of target genes through binding the octameric motif (5'-ATTTGCAT-3).
  • Oct4 functions in protein binding, miRNA binding, DNA binding, sequence-specific DNA binding, ubiquitin protein ligase binding, transcription factor activity, and transcription factor binding.
  • Oct4 is important for early embryogenesis, for embryonic stem cell pluripotency, and is required for maintaining pluripotency. Oct4 is primarily expressed in ESCs, where it is essential in maintaining potency. In later mammalian development, Oct4 expression is confined to the developing germ cells. Oct4 is conserved in human (SEQ ID NO:8), chimpanzee (SEQ ID NO:9), mouse (SEQ ID NO:10), and many other animals.
  • the SOX2 gene also known as SRY (sex determining region Y)-box2, encodes a protein that is a member of the SRY-related HMG-box (SOX) family of transcription factors.
  • SOX2 is an intronless gene that lies within an intron of another gene called the SOX2 overlapping transcript (SOX2OT).
  • SOX2OT SOX2 overlapping transcript
  • the transcription factor encoded by SOX2 forms a trimeric complex with OCT4 on DNA and controls the expression of a number of genes involved in embryonic development such as YES1, FGF4, UTF1, and ZFP206.
  • SOX2 is involved in the regulation of embryonic development, stem-cell maintenance in the central nervous system, and the determination of cell fate.
  • the SOX2 gene is conserved in human (SEQ ID NO:1), chimpanzee (SEQ ID NO:2), mouse (SEQ ID NO:3), and many other animals.
  • the Nanog gene encodes the homeobox protein Nanog. Nanog is thought to function in concert with other factors such as Oct 4 and SOX2.
  • the homeobox protein Nanog binds to the DNA consensus sequence 5'-TAAT[GT][GT]-3' or 5'-[CG][GA][CG]C[GC]ATTAN[GC]-3' (SEQ ID NO:4).
  • Nanog functions as a transcription regulator involved in inner cell mass and ESC proliferation and self-renewal. Nanog imposes pluripotency on ESC and prevents their differentiation towards extraembryonic endoderm and trophectoderm lineages. When overexpressed, Nanog promotes cells to enter into S phase and proliferate. Nanog is also associated with ovarian germ cell tumors and sacrococcygeal teratoma. The Nanog gene is conserved in human (SEQ ID NO:5), chimpanzee (SEQ ID NO:6), mouse (SEQ ID NO:7), and many other animals.
  • the Rex1 gene also called ZFP42 is expressed primarily in undifferentiated stem cells, both in the embryo and the testicles of adults.
  • the Rex1 protein is 310 amino acids long, and has four closely spaced zinc fingers at 188-212, 217-239, 245-269, and 275-299.
  • Rex1 functions in sequence-specific DNA binding transcription factor activity and is involved in ESC self-renewal.
  • the Rex1 gene is conserved in human (SEQ ID NO:11), chimpanzee (SEQ ID NO:12), mouse (SEQ ID NO:13), and many other animals.
  • the Err ⁇ gene is also known as estrogen-related receptor beta (ESRRB) and NR3B2. This gene encodes the Steroid hormone receptor ERR2 protein which is similarity to the estrogen receptor. A similar gene in mouse plays an essential role in placental development. The Err ⁇ gene is conserved in human (SEQ ID NO:50), chimpanzee (SEQ ID NO:51), mouse (SEQ ID NO:52), and many other animals.
  • TEAD4 (TEA domain family member 4) is a member of the transcriptional enhancer factor (TEF) family of transcription factors and is essential for initiating the TSC lineage. It is preferentially expressed in the skeletal muscle, and binds to the M-CAT regulatory element found in promoters of muscle-specific genes to direct gene expression. Alternatively spliced transcripts encode different isoforms. TEAD4 (TEA domain family member 4) plays a key role in the control of organ size and tumor suppression. The TEAD4 gene is conserved in human (SEQ ID NO:70), chimpanzee (SEQ ID NO:72), mouse (SEQ ID NO:71), and many other animals.
  • TEF transcriptional enhancer factor
  • the Caudal Type Homeobox 2, or Cdx2 gene is also known as Cdx3 and CDX2/AS. This gene is a member of the caudal-related homeobox transcription factor gene family.
  • the encoded protein is a major regulator of intestine-specific genes involved in cell growth and differentiation. This protein also plays a role in early embryonic development of the intestinal tract and is essential in establishing TSCs.
  • the Cdx2 gene is conserved in human (SEQ ID NO:44), Rhesus monkey (SEQ ID NO:45), mouse (SEQ ID NO:46), and many other animals.
  • the Id2 gene is also known as the cell growth-inhibiting gene 8 (GIG8), Id2A, Id2H, and bHLHb26.
  • the Id2 gene encodes for a protein called DNA-binding protein inhibitor ID-2. That protein belongs to the inhibitor of DNA binding family, members of which are transcriptional regulators that contain a helix-loop-helix (HLH) domain but not a basic domain.
  • HHL helix-loop-helix
  • This protein inhibits the functions of basic helix-loop-helix transcription factors in a dominant-negative manner by suppressing their heterodimerization partners through the HLH domains.
  • This protein plays a role in negatively regulating cell differentiation and specifically inhibits TSC differentiation in humans and mouse.
  • the Id2 gene is conserved in human (SEQ ID NO:47), chimpanzee (SEQ ID NO:48), mouse (SEQ ID NO:49), and many other animals.
  • SAPK gene is also known as JNK2, MAPK9, p54a, JNK2A, JNK2B, SAPK1a, JNK2BETA, p54aSAPK, and JNK2 ⁇ .
  • SAPK encodes the protein mitogen-activated protein (MAP) kinase 9 (MAPK9) which is a member of the MAP kinase family.
  • MAPK9 protein mitogen-activated protein
  • Several alternatively spliced transcript variants encoding distinct isoforms have been reported. The different isoforms vary in the use of an alternate internal coding exon of the same length.
  • MAP kinases act as an integration point for multiple biochemical signals, and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation, and development.
  • MAPK9 targets specific transcription factors, and thus mediates immediate-early gene expression in response to various cell stimuli.
  • MAPK9 is closely related to MAPK8, both of which are involved in UV radiation-induced apoptosis.
  • This gene and MAPK8 are also known as c-Jun N-terminal kinases. Studies of this gene's mouse counterpart suggest a key role in T-cell differentiation.
  • the SAPK gene is conserved in human (SEQ ID NO:38), chimpanzee (SEQ ID NO:39), mouse (SEQ ID NO:40), and many other animals.
  • the JNK gene also known as MAPK8, encodes the C-jun-amino-terminal kinase-interacting protein 3 enzyme. This enzyme is a member of the MAP kinase family which includes MAPK9. Five alternatively spliced transcript variants encoding distinct isoforms have been reported.
  • JNK mediates immediate-early gene expression in response to cell stress stimuli by phosphorylating specific transcription factors.
  • the activation of this kinase by tumor-necrosis factor alpha (TNF- ⁇ ) is found to be required for TNF- ⁇ induced apoptosis.
  • TNF- ⁇ tumor-necrosis factor alpha
  • This kinase is also involved in UV radiation induced apoptosis.
  • the MAPK8 gene is conserved in human (SEQ ID NO:41), chimpanzee (SEQ ID NO:42), mouse (SEQ ID NO:43), and many other animals.
  • the MEK1 gene is also known as mitogen-activated protein kinase kinase 1 (MAPKK1), CFC3, and PRKMK1.
  • MAPKK1 has been shown to interact with C-Raf, Phosphatidylethanolamine binding protein 1, MAP2K1IP1, GRB10, MAPK3, MAPK8IP3, MAPK1, MP1, and MAP3K1.
  • the protein encoded by MEK1 is a member of the dual specificity protein kinase family, which acts as a mitogen-activated protein (MAP) kinase.
  • MAP mitogen-activated protein
  • This protein kinase lies upstream of MAP kinases and stimulates the enzymatic activity of MAP kinases upon wide variety of extra-and intracellular signals.
  • MAP kinase signal transduction pathway As an essential component of the MAP kinase signal transduction pathway, this kinase regulates many processes such as proliferation, differentiation, transcription regulation, and development.
  • the MEK1 gene is conserved in human (SEQ ID NO:53), chimpanzee (SEQ ID NO:54), mouse (SEQ ID NO:55), and many other animals.
  • the MEK2 gene is also known as mitogen-activated protein kinase kinase 2 (MAPKK2), MAP2K2, CFC4, MKK2, and PRKMK2.
  • MAPKK2 mitogen-activated protein kinase kinase 2
  • MAP2K2 MAP2K2
  • CFC4 mitogen-activated protein kinase kinase 2
  • PRKMK2 PRKMK2
  • This kinase plays a critical role in mitogen growth factor signal transduction. It phosphorylates and activates MAPK1/ERK2 and MAPK2/ERK3. The activation of this kinase is dependent phosphorylation by MAP kinase kinases.
  • the MEK2 gene is conserved in human (SEQ ID NO:56), chimpanzee (SEQ ID NO:57), mouse (SEQ ID NO:58), and many other animals.
  • the AKT gene is also known as v-akt murine thymoma viral oncogene homolog 1, AKT1, PKB, PRKBA, PKB ⁇ , and RAC ⁇ .
  • AKT1 v-akt murine thymoma viral oncogene homolog 1
  • PKB PKB
  • PRKBA PKB
  • PKB ⁇ PKB
  • RAC ⁇
  • AKT is involved in regulating many processes including metabolism, proliferation, cell survival, and growth. The regulation is mediated through serine and/or threonine phosphorylation of many downstream, substrates. Over 100 substrate candidates have been reported.
  • the AKT gene is conserved in human (SEQ ID NO:59), chimpanzee (SEQ ID NO:60), mouse (SEQ ID NO:61), and many other animals.
  • the placental Lactogen gene (PL1) is also known as PL, CSH1, CSA, CS-1, CSMT, hCS-1, and hCS-A.
  • the protein encoded by this gene is a member of the somatotropin/prolactin family of hormones.
  • the PL1 gene is expressed mainly in the placenta and is produced by the 1 st lineage differentiating from mouse TSC utilizes multiple transcription initiation sites.
  • PL1 plays an important role in growth control.
  • the encoded protein is produced only during pregnancy and is involved in stimulating lactation, fetal growth, and metabolism.
  • the human PL1 gene (SEQ ID NO:62) has orthologs in chimpanzee (SEQ ID NO:63), and many other animals.
  • the Plf gene is also known as, Prl2c2 (prolactin family 2, subfamily c, member 2), Plf1, MRP-1, and PLF-1. This gene encodes the anterior pituitary hormone prolactin which is also known as luteotropic hormone or luteotropin.
  • Prl Prolactin
  • Lrp2 The protein encoded by the Lrp2 gene is called low density lipoprotein-related protein 2.
  • the Lrp2 protein is also known as.
  • Lrp2 is a multi-ligand endocytic receptor glycoprotein with a large amino-terminal extracellular domain, a single transmembrane domain, and a short carboxy-terminal cytoplasmic tail that is found in the plasma membrane of many absorptive epithelial cells..
  • Lrp2 is expressed in many different tissues but primarily in absorptive epithelial tissues such as the kidney.
  • the extracellular ligand-binding-domains of the protein bind diverse macromolecules including albumin, apolipoproteins B/ E, and lipoprotein lipase.
  • Lrp2 is important for the reuptake of numerous ligands, including lipoproteins, sterols, vitamin-binding proteins, and hormones.
  • the Lrp2 gene is conserved in human (SEQ ID NO:23), chimpanzee (SEQ ID NO:24), mouse (SEQ ID NO:25), and many and other animals.
  • the DAB2 gene was initially named DOC2 (Differentially expressed in Ovarian Cancer) and is distinct from the DOC2A and DOC2B genes. Dab2 is expressed in normal ovarian epithelial cells, but is decreased in ovarian carcinomas, suggesting a role as a tumor suppressor. The Dab2 gene is conserved in human (SEQ ID NO:20), chimpanzee (SEQ ID NO:21), mouse (SEQ ID NO:22), and many other animals.
  • the Fgf5 gene is a member of the fibroblast growth factor (FGF) family that encodes the Fibroblast Growth Factor 5 protein.
  • FGF fibroblast growth factor
  • FGF family members possess broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including embryonic development, cell growth, cell proliferation, morphogenesis, cell differentiation, tissue repair, tumor growth, and invasion.
  • This gene is as an oncogene, transforming transfected mammalian cells.
  • the Fgf5 gene is conserved in human (SEQ ID NO:32), chimpanzee (SEQ ID NO:33), mouse (SEQ ID NO:34), and many other animals.
  • the Pdgfra (platelet-derived growth factor receptor, alpha polypeptide) gene codes for the alpha-type platelet-derived growth factor receptor protein.
  • Pdgfra there is another isoform Pdgfrb.
  • the two isoforms can form homo- or heterodimers.
  • Pdgfra functions as a tyrosine-protein kinase that acts as a cell-surface receptor for Pdgfa, Pdgfb, and Pdgfc.
  • the ⁇ -type platelet-derived growth factor receptor protein plays an important role in the regulation of embryonic development, cell proliferation, survival, and chemotaxis.
  • the Pdgfra gene is conserved in human (SEQ ID NO:14), chimpanzee (SEQ ID NO:15), and many other animals.
  • the homologous gene in mice (SEQ ID NO:16) includes a protein tyrosine kinase conserved domain rather than the protein kinase domain found in the human gene.
  • the cubulin gene is also known as CUBN, IFCR, MGA1, and gp280.
  • Cubulin is located within the epithelium of intestine and kidney. Cubulin acts as a receptor for intrinsic factor-vitamin B12 complexes. Cubulin plays a role in lipoprotein, vitamin and iron metabolism, by facilitating their uptake. This gene plays an important role in the development of the peri-implantation embryo through internalization of APOA1 and cholesterol. It also binds to LGALS3 at the maternal-fetal interface.
  • the cubulin gene is conserved in human (SEQ ID NO:67), mouse (SEQ ID NO:68), and many other animals.
  • Sox17 (SRY (sex determining region Y)-box 17) gene encodes a member of the SOX (SRY-related HMG-box) family of transcription factors.
  • the encoded protein acts as a transcriptional regulator after forming a complex with other proteins. Sox17 binds target promoter DNA and bends the DNA by binding to the sequences 5'-AACAAT-'3 or 5'-AACAAAG-3'.
  • SOX17 is involved in the regulation of embryonic development and in the determination of cell fate.
  • the SOX17 gene is conserved in human (SEQ ID NO:17), chimpanzee (SEQ ID NO:18), mouse (SEQ ID NO:19), and many other animals.
  • the GATA6 (GATA binding protein 6 or transcription factor GATA-6) gene encodes a protein that is a member of a small family of zinc finger transcription factors.
  • the GATA6 protein preferentially binds (A/T/C)GAT(A/T)(A) of the consensus binding sequence.
  • GATA6 is expressed during early embryogenesis and localizes to endo- and meso-dermally derived cells during later embryogenesis and plays an important role in the regulation of cellular differentiation and organogenesis during vertebrate development such as gut, lung, and heart development.
  • the GATA6 gene is conserved in human (SEQ ID NO:26), chimpanzee (SEQ ID NO:27), mouse (SEQ ID NO:28), and many other animals.
  • GATA4 is involved in embryogenesis and in myocardial differentiation and function and testicular development. It is a transcriptional activator that binds to the consensus sequence 5'-AGATAG-3'. The GATA4 gene is conserved in human (SEQ ID NO:73), mouse (SEQ ID NO:74), and many other animals.
  • the T gene encodes the Brachury protein which is an embryonic nuclear transcription factor that binds to a specific DNA element, the palindromic T-site. Brachury binds through a region in its N-terminus called the T-box. Two transcript variants encoding different isoforms have been found for this gene.
  • the gene brachyury appears to have a conserved role in defining the midline of a bilateral organism, and thus the establishment of the anterior-posterior axis; this function is apparent in chordates and mollusks.
  • Brachury effects transcription of genes required for mesoderm formation and differentiation and is localized to notochord-derived cells.
  • the T gene is conserved in human (SEQ ID NO:29), chimpanzee (SEQ ID NO:30), mouse (SEQ ID NO:31), and many other animals.
  • the AMPK genes are also known as PRKAA1/2 and AMPKa1/2.
  • the protein encoded by this gene belongs to the ser/thr protein kinase family.
  • the functional enzyme is composed of ⁇ -(catalytic) and ⁇ / ⁇ -(regulatory) subunits.
  • the AMPK genes encode proteins called 5'-prime-AMP-activated protein kinase which is the catalytic subunit.
  • the 5'-AMP-activated protein kinase catalytic subunit ⁇ 1 has a magnesium cofactor and is 559 amino acids long with a molecular weight of 64,009 Da. Alternatively spliced transcript variants encoding distinct isoforms have been observed.
  • AMPK is a cellular energy sensor. AMPK regulates key metabolic enzymes through phosphorylation. This kinase activity is activated by stimuli that increase the AMP/ATP ratio.
  • the AMPK gene is conserved in human (SEQ ID NO:35), chimpanzee (SEQ ID NO:36), mouse (SEQ ID NO:37), and many other animals.
  • PAX8 encodes a member of the paired box (PAX) family of transcription factors involved in functional differentiation and maintenance of thyroid-related cells.
  • the PAX8 gene is conserved in human (SEQ ID NO:75), chimpanzee (SEQ ID NO:77), mouse (SEQ ID NO:76), and many other animals.
  • the NEFL gene encodes neurofilaments in the axoskeleton. They maintain neuronal caliber and also play a role in intracellular transport to axons and dendrites.
  • the NEFL gene is conserved in human (SEQ ID NO:78), chimpanzee (SEQ ID NO:80), mouse (SEQ ID NO:79), and many other animals.
  • the TSHR gene encodes membrane proteins that serve as receptors for thyrothropin and thyrostimulin. These proteins are involved in thyroid cell metabolism. Different transcript variants have been identified.
  • the TSHR gene is conserved in human (SEQ ID NO:81), chimpanzee (SEQ ID NO:83), mouse (SEQ ID NO:82), and many other animals.
  • the laminin gene encodes proteins that mediate attachment, migration and organization of cells into embryonic tissues. The proteins function by interacting with the extracellular matrix.
  • the laminin gene is conserved in human (SEQ ID NO:84), ), chimpanzee (SEQ ID NO:86), mouse (SEQ ID NO:85) and many other animals.
  • the AFP gene encodes alpha-fetoprotein, a plasma and intracellular protein produced by the yolk sac and liver during fetal development. AFP, the fetal counterpart of serum albumin and binds copper, nickel, fatty acids and bilirubin.
  • the AFP gene is conserved in human (SEQ ID NO:87), chimpanzee (SEQ ID NO:89), mouse (SEQ ID NO:88), and many other animals.
  • the NES gene encodes a member of the intermediate filament protein family and is expressed primarily in nerve cells. It is required for survival, renewal and mitogen-stimulated proliferation of neural progenitor cells.
  • the NES gene is conserved in human (SEQ ID NO:90), chimpanzee (SEQ ID NO:92), mouse (SEQ ID NO:91), and many other animals.
  • the Goosecoid (GSC) gene encodes a member of the bicoid subfamily of the paired (PRD) homeobox family of proteins.
  • the encoded protein acts as a transcription factor and plays a role in spatial programming within discrete lineage compartments during organogenesis.
  • the GSC gene is conserved in human (SEQ ID NO:93), chimpanzee (SEQ ID NO:95), mouse (SEQ ID NO:94), and many other animals.
  • Oct4 and Rex1 are markers of the 1 st ESC progenitors that arise in the embryo within 3.5 days (E3.5) after fertilization. Upon differentiation to any lineage, Rex1 is decreased (e.g., lost) and Oct4 is decreased after commitment to the 1 st differentiated lineage of extraembryonic ectoderm (ExEndo). Thus, the presence of Oct4 and Rex1 indicates stem cell potency.
  • Pdgfra is a growth factor receptor and Dab2 a lipid nutrient-acquiring protein that are both activated immediately upon 1 st differentiation of ESC to ExEndo.
  • Pdgfra is expressed in all ExEndo lineages and Dab2 only in 1 st lineage and its maturing descendants. Thus, the presence of Pdgfra and Dab2 indicate loss of stem cell potency under conditions wherein potency would normally be maintained (e.g., that a stress has occurred).
  • Sox2, Oct4 and Nanog Three embryonic genes decrease and then rapidly increase when ESCs are stressed. These 3 genes are Sox2, Oct4 and Nanog. There is a 4 th gene, Rex1, whose expression after stress decreases but does not increase thereafter as Sox2, Oct4 and Nanog do. Once expression for Rex1 decreases, this decrease indicates that stress has occurred. In CE, the stress leads to reduced stem cell potency and development potential. Within these embodiments, stress can be detected by time course evaluation of Sox2, Oct4 and Nanog expression and/or time course or single time point evaluation of Rex1.
  • the terminal differentiation of stem cells leads to production of gene products of parenchymal differentiation that distinguishes the 1 st lineage available to ESCs and TSCs, and from lineages that normally arise later from these two stem cell types.
  • the careful measure of the ratio of 1 st /later lineage intracellular and secreted products predicts stressed stem cells and pregnancies.
  • compensatory and prioritized differentiation predicts that earliest pregnancy tests will have higher or similar 1 st lineage hormone (e.g., PL1), and decreased later lineage hormones from maternal blood at the earliest times.
  • the occurrence of a stress can be detected by measuring the expression or activity of potency and/or differentiation factors that cause the up- or down-regulation of a marker.
  • the expression or activity of these factors can identify the occurrence of stress-induced compensatory and prioritized differentiation.
  • increased expression of PL1 or Plf is associated with an up-regulation of SAPK's expression or activity.
  • the continued down-regulation of Rex1 following stress is mediated by an up-regulation of JNK's expression or activity.
  • up-regulation and “down-regulation” of gene or protein expression, and protein activity can be measured against a relevant control condition including relative to the expression or activity of an unstressed stem cell or relative to differentiation mediated by removal of growth factors that maintain stemness in culture (e.g., normal differentiation control).
  • up-regulation or down-regulation will be a statistically-significant difference (e.g., p ⁇ 0.05) from a relevant control condition.
  • stem cells are assessed by measuring gene expression.
  • the stem cell parameter is a gene expression level or profile. Determining the expression of one or more genes, e.g., obtaining an expression profile) may be made by measuring nucleic acid transcripts, e.g. mRNAs, of genes of interest, e.g. a nucleic acid expression profile; or by measuring levels of one or more different proteins/polypeptides that are expression products of one or more genes of interest, (e.g. a proteomic expression profile).
  • expression profile and “expression evaluation” can be used broadly to include a gene expression profile at the nucleic acid level or a gene expression profile at the protein level.
  • expression of genes may be evaluated by obtaining a nucleic acid expression profile, where the amount or level of one or more nucleic acids in a sample is determined.
  • the nucleic acid sample includes a plurality or population of distinct nucleic acids that includes the expression information of the genes of interest of the stem cell being assessed.
  • the nucleic acid may include RNA or DNA nucleic acids, e.g., mRNA, cRNA, cDNA etc., so long as the sample retains the expression information of the stem cell from which it is obtained.
  • the sample may be prepared in a number of different ways, e.g., by mRNA isolation from a cell, where the isolated mRNA is used as is, amplified, employed to prepare cDNA, cRNA, etc., as performed in the differential expression art.
  • the sample may be prepared from a single cell, e.g. a stem cell or a population of stem cells of interest.
  • the expression profile may be generated from the initial nucleic acid sample using many appropriate protocols.
  • One representative protocol is array-based gene expression profile generation protocols.
  • Such protocols can be hybridization assays in which a nucleic acid that displays "probe" characteristics for each of the genes to be assayed/profiled in the profile to be generated is employed.
  • an array of "probe" nucleic acids that includes a probe for each of the phenotype determinative genes whose expression is being assayed is contacted with target nucleic acids. Contact is carried out under hybridization conditions, e.g., stringent hybridization conditions, and unbound nucleic acid is then removed.
  • hybridization conditions e.g., stringent hybridization conditions
  • stringent hybridization conditions include e.g., 2 hr to 4 days incubation at 42°C using a DIG-labeled DNA probe (prepared by, e.g., using a DIG labeling system; Roche Diagnostics GmbH, 68298 Mannheim, Germany) in a solution such as DigEasyHyb solution (Roche Diagnostics GmbH) or a solution including 50% formamide, 5XSSC (150 mM NaCl, 15 mM trisodium citrate), 0.02% sodium dodecyl sulfate, 0.1% N-lauroylsarcosine, and 2% blocking reagent (Roche Diagnostics GmbH), followed by washing the filters twice for 5 to 15 minutes in 2XSSC and 0.1% SDS at room temperature and then washing twice for 15-30 minutes in 0.5XSSC and 0.1% SDS or 0.1XSSC and 0.1% SDS at 65-68°C.
  • a DIG-labeled DNA probe prepared by, e.g., using a DIG
  • Probes can also be designed to have a % sequence identity that is complementary to wild-type gene sequences that lead to conditions that are compatible to produce binding pairs of nucleic acids, e.g., surface bound and solution phase nucleic acids, of sufficient complementarity to provide for the desired level of specificity in the assay while being less compatible to the formation of binding pairs between binding members of insufficient complementarity to provide for the desired specificity.
  • the probes will have 90% to 100% sequence identity in 1% stepwise increments (e.g., 90%, 91%, 92% up to 100%) that identity a sequence that is complementary to the wild-type sequence being assessed.
  • % sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between sequences as determined by the match between strings of such sequences.
  • Identity (often referred to as “similarity") can be readily calculated by appropriate methods, including those described in: Computational Molecular Biology (Lesk, ed.) Oxford University Press, NY 1988 ; Biocomputing: Informatics and Genome Projects (Smith, ed.) Academic Press, NY 1994 ; Computer Analysis of Sequence Data, Part I (Griffin and Griffin, eds.) Humana Press, NJ 1994 ; Sequence Analysis in Molecular Biology (Von Heijne, ed.
  • Resultant patterns of hybridized nucleic acid provides information regarding expression for each of the genes that have been probed, where the expression information is whether or not the gene is expressed and, typically, at what level, where the expression data or expression profile (e.g., in the form of a transcriptome), may be both qualitative and quantitative.
  • non-array based S/M for quantitating the level of one or more nucleic acids in a sample may be employed, including those based on amplification protocols, e.g., Polymerase Chain Reaction (PCR)-based assays, including quantitative PCR, reverse-transcription PCR (RT-PCR), real-time PCR, etc.
  • PCR Polymerase Chain Reaction
  • RT-PCR reverse-transcription PCR
  • real-time PCR etc.
  • expression of genes may be evaluated by obtaining a proteomic expression profile, where the amount or level of one or more proteins/polypeptides in the sample is determined.
  • proteomic expression profile i.e. a profile of one or more protein levels in a sample
  • any convenient protocol for evaluating protein levels may be employed wherein the level of one or more proteins in the assayed sample is determined. While a variety of different manners of assaying for protein levels can be used, one representative protocol is enzyme-linked immunosorbent assays (ELISA).
  • non-ELISA based-S/M for measuring proteins levels in a sample such as mass spectrometry, proteomic arrays, xMAP TM microsphere technology, flow cytometry, western blotting, and immunohistochemistry can be used.
  • modified stem cells which are not part of the invention
  • reporter transgene systems that are used to identify compounds or other potential environmental stem cell stressors that cause forced, compensatory or prioritized differentiation and reduced development potential.
  • reporter genes include a nucleic acid sequence encoding a reporter.
  • the reporter transgene is inserted via homologous recombination into the stem cell genome so that the reporter gene is in operable combination with the promoter of a factor or marker (e.g., potency or differentiation) of interest.
  • “In operable combination” refers to the linkage of nucleic acid sequences in a manner such that one or more proteins can be produced.
  • a promoter is a regulatory element that facilitates the initiation of transcription of a coding region of a nucleic acid sequence in operable combination (used interchangeably with operably linked) with it.
  • a promoter is typically, though not necessarily, located 5' (i.e., upstream) of a nucleotide sequence of interest whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription. With this approach, when a factor or marker gene can be expressed, it is expressed as a reporter.
  • a “gene” refers to a nucleotide sequence that encodes a protein (e.g., a reporter protein or other proteins described herein). This definition includes various sequence polymorphisms, mutations, and/or sequence variants. In particular embodiments, the sequence polymorphisms, mutations, and/or sequence variants do not affect the function of the encoded protein.
  • Nucleic acid sequences encoding proteins can be DNA or RNA that directs the expression of the protein or RNA. These nucleic acid sequences may be a DNA sequence that is transcribed into RNA or an RNA sequence that is translated into protein. The nucleic acid sequences include both the full-length nucleic acid sequences as well as non-full-length sequences derived from the full-length protein or RNA. The sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference. In addition to particular sequences provided, gene sequences to encode reporter proteins and other proteins are available in publicly available databases and publications.
  • a gene of interest encodes any of the potency factors, potency markers, compensatory or prioritized differentiation factors or markers disclosed herein.
  • a reporter gene encodes any reporter disclosed herein.
  • the disclosure is not limited to the use of any particular reporter gene. Indeed, the use of a variety of reporters can be used.
  • the disclosure provides S/M for transfecting stem cells with reporter genes encoding fluorescent proteins, wherein the expression of the fluorescent proteins provides an expression assay in stem cells without having to sacrifice the cells.
  • Fluorescent reporters are known and can be obtained commercially at, e.g., Promega Corporation, Invitrogen, Clontech, Stratagene, BD Biosciences Pharmingen, or Evrogen JSC. Additional examples of fluorescent proteins can be found at, e.g., US Patent No. 6,884,620 and 6,645,761 .
  • the disclosure is not limited by the fluorescent protein-encoding gene, and any fluorescent encoding gene can be used.
  • the gene encoding the fluorescent protein can be optimized for mammalian expression by codon optimization for mammalian translational machinery.
  • the disclosure provides S/M for transfecting stem cells with reporter genes encoding enzymatic proteins, wherein the expression of the enzymatic proteins provides a viable expression assay without sacrificing the stem cells.
  • the expression of reporter enzymatic proteins provide light output upon substrate utilization. Examples of such enzymatic proteins that produce light as a byproduct of substrate utilization include, luciferase and ⁇ -lactamase. Luciferase encoding reporter genes (e.g. Photinus sp. (GenBank Accession No. AY738222), Renilla sp.
  • modified stem cells may be transfected with a vector including a wild-type promoter or a synthetic promoter made up of repeated response elements to a given single transcription factor (as done with Rex1 and Oct4 in Li (2015).
  • Rapid development and validation of a Rex1 promoter-RFP (red fluorescent reporter) viable potency activity reporter quantifies stress-induced ESC potency loss and supports the theory of compensatory differentiation.
  • the vector will integrate into the stem cell genome and on-going cellular events will interact with the inserted promoter. When conditions occur that would cause transcription of the genes associated with the wild-type promoter, expression of the inserted reporter will also occur.
  • Non-limiting examples of viral vectors that can be used include adenoviral vectors and retroviral vectors.
  • Adenoviral vectors include those constructs containing adenovirus sequences sufficient to (a) support packaging of a construct and (b) express a cell-specific construct that has been cloned therein.
  • Retroviral vectors integrate their genes into stem cell genomes and are capable of transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types ( Miller, Am. J. Clin.
  • Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. See, e.g., Naldini, Science, 272(5259):263, 1996 ; Zufferey, Nat. Biotechnol., 15(9):871, 1997 ; US Pat. Nos. 6,013,516 and 5,994,136 ).
  • Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1ad nHIV-.
  • Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, e.g., the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
  • Recombinant lentiviral vectors are capable of infecting stem cells and can be used for ex vivo gene transfer and expression of nucleic acid sequences.
  • recombinant lentivirus capable of infecting cells wherein a suitable host cell is transfected with ⁇ vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in US Pat. No. 5,994,136 .
  • Promoters used in embodiments described herein may be "endogenous” (e.g., wild-type), “exogenous” or “heterologous.”
  • An “endogenous” promoter is one that is naturally operably linked with a given gene in the genome.
  • An “exogenous” promoter is one that is not native to the wild-type genome.
  • a “heterologous” promoter is a type of exogenous promoter that shares at least 90% sequence identity with an endogenous promoter.
  • reporter gene constructs include a selectable marker.
  • the disclosure is not limited to the use of any particular selectable marker.
  • An exemplary selectable marker includes dominant selectable markers such as the bacterial aminoglycoside 3' phosphotransferase gene (also referred to as the neo gene) that confers resistance to the drug G418 in mammalian cells.
  • Other selectable markers are used in stem cell lines that lack the relevant enzyme activity.
  • non-dominant selectable markers include the thymidine kinase (tk) gene that is used in conjunction with tk-deficient cell lines and the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) gene, which is used in conjunction with hprt-deficient cell lines.
  • tk thymidine kinase
  • hprt mammalian hypoxanthine-guanine phosphoribosyl transferase
  • Gene constructs including vectors and reporter genes described herein can be inserted or "knocked-in" at a desired locus in a stem cell.
  • Methods for generating gene constructs for use in generating knock-in cell lines and techniques for generating such cell lines are described in, e.g., Sambrook 1989, Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory ; Yoo, Neuron, 37:383, 2003 ; and Lin, Human Molecular Genetics, 10:137, 2001 .
  • Exemplary knock-in methods include ex vivo transfection (see, e.g., Wilson, Science, 244:1344, 1989 ; and Nabel & Baltimore, Nature, 326:711, 1987 ), optionally with Fugene6 (Roche) or Lipofectamine (Invitrogen); injection (see, e.g., US Pat. Nos. 5,994,624 ; 5,981,274 ; 5,945,100 ; 5,780,448 ; 5,736,524 ; 5,702,932 ; 5,656,610 ; 5,589,466 ; and 5,580,859 ); microinjection (see, e.g., Harland and Weintraub, J. Cell. Biol., 101:1094, 1985 ; US Pat.
  • brake pedal/potency and accelerator pedal/differentiation constructs and modified viable reporter ESCs can be described as follows: multimerized Oct4 promoters and multimerized Rex1 promoters that drive green fluorescent protein (GFP) and red fluorescent protein (RFP) respectively can be expressed in living ESCs. Following exposure to stress, the Rex1 potency loss should be greater and more permanent than Oct4 loss. These results can be determined by immunoblots for biochemical measures and FACS and microplate reader for visual assays. Microplate readers also provide for HTS for various stresses.
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • Modified ESCs with a GFP knocked into one of the endogenous Pdgfra loci differentiation reporters have been tested.
  • GFP expression is driven by the endogenous promoter as it is activated by normal or stressed mechanisms.
  • the specificity of Pdgfra-GFP induction can be validated using other co-markers of 1 st lineage induced during stressed differentiation and later lineages that are induced during growth factor removal but suppressed during stressed differentiation.
  • DAB2, LRP2, and GATA6 are appropriate co-markers of 1 st lineage
  • ExEndo, FGF5, and SPARC are appropriate markers of other later lineages suppressed by stress-induced differentiation but activated during normal differentiation by removing potency-maintaining growth factors.
  • CE of ESC reporter cell lines of the disclosure include: (1) Potency measuring viable reporters (decreased with stress): (a) Oct4 multimerized promoter-GFP ("Oct4-GFP”); (b) Rex1 multimerized promoter-RFP ("Rexl-FRP”); (c) Oct4-GFP/Rex1-RFP co-expressing ESCs ("Oct4-GFP/Rex1-RFP”); (2) Differentiated 1 st lineage measuring viable reports (increased with stress): (a) Pdgfra promoter-GFP knockin ("Pdgfra-GFP”); (b) Sox17 Endo promoter-GFP knockin ("Sox17-GFP”); (c) DAB2, LRP2 Endo promoter-GFP; (d) GATA6 Endo promoter-GFP; (3) Later lineage measuring promoters (decreased with stress): (a) Brachyury Endo promoter-GFP knockin (“Brachy-GFP”);
  • modified viable reporters such as PL1 promoter-RFP can be used to measure transcription factors marking the differentiation to 1 st lineage available to TSCs.
  • CE of the disclosure utilizing Rex1-RFP for brake pedal, potency-loss testing and Pdgfra-GFP for accelerator pedal, 1 st lineage differentiation-increase testing represent preferred viable ESC-HTS reporters. Because submorbid, runting doses that cause differentiation are used, it is important, and technically feasible to add a nuclear stain to the color plate reader excitation and band pass acquisition and assay the level of diminished stem cell population expansion. At any stress dose, taken together or individually (i) decreased potency, (ii) increased 1 st lineage, and (iii) diminished cell population expansion should give an accurate analysis of stress impacts on embryonic and fetal development in response to toxicological stress and is completely unique amongst stem cell assays for toxicology.
  • a sample is assayed to generate an expression profile that includes expression data for at least 1 gene/protein, sometimes a plurality of genes/proteins, where by plurality is meant at least 2 different genes/proteins, and often at least 3, at least 4, at least 5, or at least 10 different genes/proteins, etc.
  • the Luminex Xmap system (Luminex Corp, Austin, TX) allows for, e.g., 500 simultaneous fluorescent frequency multiplexing.
  • the expression evaluation may be qualitative or quantitative.
  • the S/M provide an assessment, of whether or not the target analyte is present in the sample being assayed.
  • the S/M provide a quantitative detection of whether the target analyte is present in the sample, e.g., an assessment of the actual amount or relative abundance of the target analyte
  • S/M can include comparisons to reference levels.
  • Reference levels can be obtained from a dataset.
  • a reference level from a dataset can be derived from previous measures derived from a population.
  • a "population" is any grouping of stem cells of like specified characteristics. The grouping could be according to, e.g., previous exposure to a stress condition or lack thereof.
  • a “dataset” as used herein is a set of numerical values resulting from evaluation of a sample (or population of samples) under a desired condition.
  • the values of the dataset can be obtained, e.g., by experimentally obtaining measures from a sample and constructing a dataset from these measurements.
  • the reference level can be based on e.g., any mathematical or statistical formula useful in the art for arriving at a meaningful aggregate reference level from a collection of individual datapoints; e.g., mean, median, median of the mean, etc.
  • a reference level or dataset to create a reference level can be obtained from a service provider such as a laboratory, or from a database or a server on which the dataset has been stored.
  • CE conclusions are drawn based on whether a measure is statistically significantly different or not statistically significantly different from a reference level.
  • a measure is not statistically significantly different if the difference is within a level that would be expected to occur based on chance alone. In contrast, a statistically significant difference or increase is one that is greater than what would be expected to occur by chance alone.
  • Statistical significance or lack thereof can be determined by any of various S/M used in the art.
  • An example of a commonly used measure of statistical significance is the p-value. The p-value represents the probability of obtaining a given result equivalent to a particular datapoint, where the datapoint is the result of random chance alone. A result is often considered significant (not random chance) at a p-value less than or equal to 0.05.
  • Embodiments can be used with high throughput screening (HTS).
  • HTS high throughput screening
  • stem cells e.g., modified stem cells
  • a microtiter plate including, e.g., a 96- or 384-well plate that can be read in a fluorescent microplate reader.
  • the stem cell lines in the wells can then be exposed to different compounds and/or different concentrations of compounds.
  • stem cells can be contacted with a single test agent, or, to facilitate HTS, to at least 2, at least 5, at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000 or at least at least 1,000,000 test compounds at a time.
  • Environmental conditions can similarly be screened, and test compounds and environmental conditions can be screened in various combinations. If the stem cells score positive for stress, a different set of stem cells can be subsequently tested with a subset of the test compounds or environmental conditions until the compounds or conditions creating the stress effect are identified.
  • High-throughput screening systems are commercially available from, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick, Mass., Luminex Corp., etc.. These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for the various S/M of high throughput.
  • Kits to practice the S/M are also provided (which are not part of the invention).
  • Kits can include one or more containers including modified stem cells, primer pairs, probe sequences, binding proteins (e.g., antibodies) and/or reagents or supplies to assess expression of a gene of interest described herein.
  • Components of the kit can be prepared for storage and later use. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of the kit, which notice reflects approval by the agency of manufacture, use, or sale, when required.
  • kits further include instructions for using the kit in the methods.
  • the instructions can include appropriate reference levels to interpret results associated with using the kit; proper disposal of the related waste; and the like.
  • the instructions can be in the form of printed instructions provided within the kit or the instructions can be printed on a portion of the kit itself. Instructions may be in the form of a sheet, pamphlet, brochure, CD-Rom, or computer-readable device, or can provide directions to instructions at a remote location, such as a website.
  • Example 1 As an embryo implants into the uterus, stress can diminish placental TSCs proliferation inducing differentiation to create more essential differentiated product/cells. Xie, 2014, Xie, Int. Rev. Cell. Mol. Biol., 287:43, 2011 (Xie, 2011); Awonuga Mol. Reprod. Dev., 78:519, 2011 (Awonuga, 2011); Zhong, Reproduction, 140:921, 2010 (Zhong, 2010). This "compensatory" differentiation thus further depletes the size of the population of multipotent TSC. Most human embryos are lost before birth and the greatest loss occurs soon after implantation. Cross, Science, 266:1508, 1994 . Understanding the cellular and molecular mechanisms of TSC stress responses informs strategies to ameliorate their consequences during implantation.
  • Implantation site O 2 is normally 2%, when O 2 levels are optimized, highest growth and least stress (activation of stress-activated protein kinase/SAPK) is at 2% O 2 . Zhou, 2011. Also, dose responses of SAPK activon in response to TNFalpha, dioxin suggest that these stressors could activate compensatory and prioritized differentiation in TSC ( Xie, 2008, Zhong, Reprod. Sci., 14:534, 2007 (Zhong, 2007)). Cigarette smoke condensate and IFNgamma also may activate compensatory differentiation through Rex1 loss in mouse ESCs.
  • Fibroblast growth factor (FGF)4 is synthesized in the implanting embryo (Xie 2011) and is necessary for maintaining TSC proliferation and potency in culture (Tanaka, 1998) and in the embryo (Chai, 1998). To better emulate preimplantation stress effects, TSC culture was performed in this Example for 24 hr in the presence of FGF4. Furthermore, to study the longer period of postimplantation development, culture was extended to 7 days with FGF4 removed to stimulate normal differentiation.
  • the 1 st lineage differentiating from TSC is parietal trophoblast giant cells (pTGC) at E4.5 (4.5 days after fertilization), followed by spongiotrophoblasts at E7.0 and labyrinthine, chorionic syncytiotrophoblasts (synT) and sinusoidal TGC (sTGC) at E7.5.
  • pTGC parietal trophoblast giant cells
  • spongiotrophoblasts at E7.0 and labyrinthine
  • synT chorionic syncytiotrophoblasts
  • sTGC sinusoidal TGC
  • SAPK will mediate induction of the 1 st lineage/marker (pTGC/Hand1) and suppression of later lineage/marker (synTA/Gcm1) induced by hypoxic stress.
  • the mitochondrial membrane potential ( ⁇ m ) stains tetramethylrhodamine methyl ester (TMRM), MitoTracker red, and 5,5',6,6'-tetrachloro-1,1',3,3'-Tetra-ethylbenzimidazolylcarbo-cyanine iodide (JC1) were purchased from Molecular Probes (Grand Island, NY). Mitochondrial inhibitors antimycin A and cyanide, the mitochondrial activator dichloroacetate (DCA), and the OxPhos uncoupler Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) were purchased from Sigma (St. Louis, MO). FGF4 and heparin were from Sigma Chemical Co.
  • mice TSC Cell lines and culture conditions.
  • the mouse TSC sources and culture conditions were described previously. Awonuga, 2011; Zhou, 2011. Briefly, mouse TSC isolated from E3.5 and E6.5 conceptuses were from Dr. Rossant (Lunenfeld Research Institute, Ontario, Canada) and cultured. Cells were cultured with premixed gas at 0.5%, 2% (O 2 /CO 2 /N 2 levels of 0.5/4.5/95, and 2/5/93, respectively, Praxair, Warren, MI), and 20% O 2 levels were obtained by mixing 5% CO 2 from a 100% CO 2 gas tank with ambient N 2 and O 2 levels. Media were pre-equilibrated under O 2 levels for TSC culture overnight prior to culture.
  • Quantitative removal of gas phase O 2 requires 30 min in anaerobic bags, i.e., there was only a minimal period when 20% O 2 was ambient to media that was pre-equilibrated under 0, 0.5, or 2% O 2 . Since 12-24 hr is required to equilibrate gas phase to complex media/liquid phase, there was no appreciable exposure of TS cells to 20% O 2 again after the switch to 0, 0.5, or 2% O 2 .
  • RNA isolation and quantitative reverse transcription-PCR qRT-PCR. All steps in RNA isolation, cDNA synthesis and qPCR performance and analysis were described previously (Zhou, 2011). Briefly, DNase-treated total RNA was isolated following cDNA preparation using QuantiTect Reverse Transcription Kit iScript (Qiagen). Assays were performed using Fast SYBR Green on a System 7500 instrument (Applied Biosystems), and a one-step program for 40 cycles. Each experiment was carried out in triplicate, and all three genes for TSC multipotency were normalized against 18S rRNA.
  • TSC Mitochondrial staining by MitoTracker dye.
  • TSC were cultured at different O 2 levels at least 24 hr stained with DAPI (D21490, Invitrogen) alone or with either MitoTracker Red FM (Invitrogen), TMRM (T668), or JC-1 (T3168, Invitrogen) alone or with the uncoupler FCCP (Sigma), and analyzed under a DM-IRE2 fluorescence microscope (Leica, Germany) using Simple PCI image acquisition software (Hamamatsu, Sewickley, PA).
  • TSC were cultured on coverslips in 12-well plates at different O 2 levels either with or without FGF4, for 48 hr.
  • TSC were adapted in a hypoxia chamber (Billups-Rothenberg, CA) at the given O 2 level described above for at least 24 hr.
  • DAPI Nuclear dye 25 ⁇ g/ml DAPI (D21490, Invitrogen) was applied as a counterstain 1 hr before TSC were incubated alone with either 50nM MitoTracker Red FM (M22425m, Invitrogen) for 30 min, 20nM TMRM (T668) for 30 min, or 3.25nM JC-1 (T3168, Invitrogen) for 45 min, or in combination with the uncoupler FCCP (1nM, Sigma, C2920). After staining, the TSC were washed with PBS 2 times and immediately analyzed under a DM-IRE2 fluorescence microscope (Leica, Germany) using Simple PCI image acquisition software (Hamamatsu, Sewickley, PA).
  • ATP measurements TSC were washed, collected, centrifuged, frozen on dry ice, and stored. Cell pellets were boiled, lysed, iced, and solubilized by ultrasonication. (ATP)s were determined using the ATP bioluminescence assay kit HS II (Roche Applied Science) by the manufacturer's protocol. Data were standardized to the protein concentration. In more detail, TSC were washed with PBS once, collected by scraping, centrifuged at 10,000 rpm for 30 sec and then PBS was quickly removed. Cells were immediately frozen on dry ice and stored at - 80°C.
  • ATP concentrations were determined using the ATP bioluminescence assay kit HS II (Roche Applied Science) according to the manufacturer's protocol. Data were standardized to the protein concentration.
  • ROS measurements at 20% O 2 TSC were trypsinized, centrifuged, washed, and incubated with the ROS-sensitive probe 2' ,7' -dichlorodihydrofluorescein diacetate (Molecular Probes), and analyzed on an Ascent Fluoroskan plate reader. Experiments were performed in triplicate and data standardized to cell number. As a control, cells were incubated in the presence of 100 ⁇ M H 2 O 2 .
  • TSC were trypsinized, collected by centrifugation, washed, and incubated with the ROS-sensitive probe 2' ,7' - dichlorodihydrofluorescein diacetate (H 2 DCFDA; D-399, Molecular Probes) at a final concentration of 7.5 ⁇ M for 20 min at 37°C in the dark.
  • H 2 DCFDA ROS-sensitive probe 2' ,7' - dichlorodihydrofluorescein diacetate
  • Cells were collected by centrifugation, supplemented with media, and incubated in the dark for 20 min at 37°C.
  • As a control cells were incubated in the presence of 100 ⁇ M H 2 O 2 .
  • ETC activity is a product of changes with FGF4 removal of total protein (dependent on O 2 levels) multiplied by the change in posttranslational regulation (dependent on FGF4) and this product is multiplied by the total protein levels after FGF4 removal that is acted upon.
  • SAPK increases the 1 st lineage and decreases later lineages proportional to the deviation from the optimal O 2 at 2%.
  • Two SAPK inhibitors with different mechanisms, SP600125 and LJNKI1 were used to limit possible off-target effects. Both SAPK inhibitors prompted a 3-7-fold increase in the later chorionic lineage (Gcm1), with the highest inhibition occurring at 0.5% and 20% O 2 ( FIGs. 1A and 2 ). Both SAPK inhibitors also increased later spongiotrophoblast lineage (Tpbpa) at all O 2 levels. In contrast, both inhibitors suppressed early lineage pTGC (Hand1) mRNA ( FIGs. 1A and 2 ) with suppression being highest at 0.5% and 20% O 2 .
  • both SAPK inhibitors suppressed later lineages (Tpbpa and Gcm1) and increased the earliest lineage (Hand1, FIG. 1B and FIG. 2 , 3A and 3B ).
  • the smallest effects on Hand1, Tpbpa, and Gcm1 occurred at 2% O 2 , but as O 2 levels deviated from 2% the mRNA levels are influenced by SAPK level upward for Hand1 and downward for Gcm1 ( FIG. 1B ).
  • Loss of potency and gain of differentiation are O 2 -dependent, and hypoxia suppresses labyrinthine placenta. It was 1 st tested whether the most stressful 0.5% O 2 level induces most loss of potency factor transcripts and increased differentiation after FGF4 removal. It was found that following FGF4 removal, the caudal homeobox related (Cdx2), Inhibitor of Differentiation (ld)2, and estrogen receptor related (Err) ⁇ - potency-mediating mRNA - were the most rapidly lost by 24 hr at 0-0.5% O 2 ( FIG. 4A , 2 ). In contrast by day 7 0.5% O 2 sustained the highest potency factor mRNA levels while the lowest average level of the potency factors were observed at 20% O 2 .
  • Cdx2 caudal homeobox related
  • ld Inhibitor of Differentiation
  • Err estrogen receptor related
  • Proliferin (Plf), placental lactogen (PL)1, CtsQ, and synTa had a maximal increase of 7-10 fold, and 1 marker (placental lactogen, (PL)2) had a 70-fold increase by day 7 at 20% O 2 ( FIG. 4B , C ).
  • the two placental surface lineages, sTGC and synTA cells appear least supported by O 2 at ⁇ 20%. Markers of these two lineages were suppressed by hypoxic stress at 0.5% O 2 to 10% of levels at 20% O 2 ( FIG. 4C ).
  • Hyperosmolar stress that induces differentiation at 20% O 2 cannot induce differentiation at hypoxic 0.5% O 2 .
  • O 2 at 0.5% does not support differentiation but also does not activate as much SAPK as 100 mM sorbitol at >20% O 2 6 .
  • 100 mM sorbitol, which increases SAPK 5 fold and induces differentiation at 20% O 2 despite FGF4 was added.
  • the added stress did not increase mRNA expression of any of the 3 terminal differentiation markers (PL1, Plf, and synTa) during hypoxia ( FIG. 6A ).
  • 0.5% O 2 cannot support differentiation after FGF4 removal or sorbitol addition.
  • Multipotent TSCs that differentiated for 2 days after FGF4 removed showed increased mitochondrial membrane potentials ( ⁇ ⁇ m ).
  • the roles of FGF4 and O 2 on ⁇ ⁇ m associated with ATP production was determined.
  • TSCs were incubated for 2 days with or without FGF4 and stained with the ⁇ ⁇ m -sensitive probes MitoTracker ( FIG. 7A-7D ), JC1, or TMRM in the presence or absence of the ⁇ ⁇ m uncoupler FCCP ( FIG. 8A-8F ).
  • MitoTracker FIG. 7A-7D
  • JC1 JC1
  • TMRM TMRM
  • FIG. 8A-8F the ⁇ ⁇ m uncoupler FCCP
  • FGF4 maintained mitochondria with low ⁇ ⁇ m .
  • Some cells had high ⁇ ⁇ m at epithelial edges despite FGF4. Similar to the MitoTracker stain findings ( FIG.
  • both JC1 and TMRM stains showed increased ⁇ ⁇ m levels only after FGF4 removal ( FIG. 8A-8F ).
  • FCCP completely depolarizes the A ⁇ m , showing the specificity of the ⁇ ⁇ m effects.
  • FGF4 maintains mitochondrial inactivity in TSCs at all O 2 levels and O 2 does not play a role in mitochondrial initiation to full activity.
  • FGF4 and hypoxia co-regulate low ⁇ ⁇ m , but ⁇ ⁇ m does not increase with FGF4 removal during hypoxia after 7 days.
  • the JC1 stain was used to assay ⁇ ⁇ m and to test whether hypoxia limited ⁇ ⁇ m generation when differentiation was maximal after FGF4 removal for 7 days (as in FIG. 4A-4D ). There were 4 levels of JC1 stain color and intensity. FCCP produced no fluorescence, indicating complete ⁇ ⁇ m depolarization ( FIG. 6B ).
  • TSCs cultured with FGF4 at 0.5% O 2 showed a low level of green fluorescence, indicating low ⁇ ⁇ m , but TSCs cultured at 2% or 20% O 2 with FGF4 removed showed two levels of increasing red fluorescence, indicating higher ⁇ ⁇ m .
  • FGF4 and hypoxia play additive, regulatory roles for ⁇ ⁇ m but even with FGF4 removed, 0.5% O 2 could not sustain high ⁇ ⁇ m levels.
  • hypoxia induced morphological changes At 0.5% O 2 , stress significantly increases nuclear size despite FGF4 and nearly all TSCs with small nuclei were lost and all differentiated cells were TGC ( FIG. 6B , 6C ).
  • Multinuclear synTA cells and giant cells were visible at 20% O 2 with FGF4 removed, but synTA cells were not observed or extremely rare at 0.5% O 2 .
  • hypoxic stress initiates differentiation to TGC morphologically while suppressing syncytiotrophoblasts
  • terminal differentiation does not complete at 7 days ( FIG. 4B ) and size of TGC is lower at 0.5% ⁇ 20% O 2 .
  • FGF4 is most important in suppressing ⁇ ⁇ m when O 2 is ⁇ 2%, but O 2 ⁇ 0.5% limits ⁇ ⁇ m generation, and morphological differentiation of TSCs is limited at 0-0.5% O 2 .
  • Cytochrome c oxidase (COX) and pyruvate kinase embryonic form M2 (PKM2) are co-regulated by FGF4 and hypoxia, and their total protein levels do not change during hypoxia despite FGF4 removal. Without being bound by theory, a likely mechanism for decreased differentiation at 0.5% O 2 is insufficient mitochondrial OxPhos, which normally consumes >90% of cellular O 2 .
  • COX is the terminal, proposed rate-limiting enzyme of the electron transport chain (ETC). Villani, J. Biol. Chem., 273:31829, 1998 ; Pacelli, Mitochondrion, 11:334, 2011 .
  • FGF4 maintained phosphorylation of PKM2 on Tyr105 at all O 2 concentrations (compare FIG. 9A and 9C ). However, for PKM2, O 2 ⁇ 2% without FGF4 resulted in the highest protein levels, with less PKM2 protein at O 2 >2% ( FIG. 9E ). At 20% O 2 , PKM2 protein decreased after FGF4 removal. The adult splice variant, PKM1, was higher with 2-20% O 2 , but there was no coherent pattern of regulation by FGF4 or O 2 ( FIG. 9D ). Despite FGF4 removal, hypoxic O 2 at 0.5% O 2 maintains high glycolysis by maintaining high PKM2 protein levels.
  • Mitochondrial antagonists and an agonist show that mitochondrial function is necessary and sufficient to mediate the differentiation of two labyrinthine placental lineages.
  • 2 mitochondrial inhibitors and an agonist were used.
  • the mitochondrial activator dichloroacetate (DCA) was tested at 0.5%-20% O 2 ( FIG. 10A ). There were no effects at 0.5% or 20% O 2 . But at 2% O 2 , of 5 markers only 2 markers increased 2-fold with DCA treatment; PL2 and synTa. These are markers of sTGC and synTA cells of the labyrinthine placenta, although PL2 is detected in other cell types.
  • Cyanide and antimycin A inhibit COX (part of mitochondrial complex IV) and complex III, respectively, and were used to inhibit the ETC and thus the generation of ⁇ ⁇ m for 7 days of culture at optimal differentiation conditions ( FIG. 10B ).
  • Sublethal concentrations of inhibitors were used that had significant effects on mitochondria.
  • both mitochondrial inhibitors decreased mRNA levels. Markers emphasized (PL2; inhibited 98% and 56%) or specific (Ctsq; inhibited 81% and 71%) for labyrinthine sTGCs were decreased greatly by antimycin and cyanide respectively ( FIG. 10B ).
  • a marker specific for labyrinthine synTA cells (synTa; inhibited 42% and 34%) was inhibited by antimycin and cyanide respectively ( FIG. 10B ).
  • mitochondrial antagonists block differentiation of 2 lineages of the labyrinthine placenta under conditions mediating maximal differentiation, whereas a mitochondrial agonist increased those lineages at 2% O 2 .
  • ATP decreases in an O 2 -dependent manner as TSCs differentiate, but highest ATP accumulation occurs where hypoxic differentiation is lowest.
  • TSCs were cultured for 1-7 days without FGF4, and ATP levels were determined on days 1, 2, 4, and 7, the same as those used to test differentiation ( FIG. 4B , 4C ).
  • ATP continuously decreased from days 1-7, with the lowest ATP in TSCs cultured at 2% or 20% O 2 at day 7 ( FIG. 11 ).
  • energy levels were lowest when differentiation was highest.
  • FGF4 suppresses ROS generation by TSCs.
  • FGF4 regulates mitochondrial function through COX1 and PKM2 phosphorylation, which reduces ETC by preventing carbon entry into mitochondria.
  • Mitochondria produce ROS, which can endanger DNA and are suppressed in stem cells. Simon and Keith, Nat. Rev. Mol. Cell Biol., 9:285, 2008 .
  • TSCs produce twice as much ROS (H 2 O 2 ) 48 hr after FGF4 removal ( FIG. 12 ). Thus FGF4 maintains low ROS.
  • hypoxia-induced hand1 mediates differentiation of the 1 st lineage, pTGC.
  • a key problem during the hypoxic stress at 0-0.5% O 2 is that it induces TSC differentiation in the 1 st 24-48 hr increase, marked by increases in cell size and PL1 and Plf levels (early lineage markers), but these markers are less than at 20% O 2 between days 4-7 due to mitochondrial insufficiency.
  • the tendency is that 1 st lineage markers, which are favored early with hypoxic stress here and by all stresses at 20% O 2 as reported here and previously (Awonuga, 2011; Rappolee, Syst. Biol. Reprod. Med., 58:33, 2012 ), have a low "ceiling" of differentiation at 0.5% O 2 after 7 days with or without sorbitol. This suggests that energy or another mitochondrial function is rate-limiting.
  • both mitochondrial inhibitors decrease all differentiated lineages but the largest effects are on the labyrinthine surface lineages. But the highest inhibition occurs in the order 20>2>>0.5% O 2 after FGF4 removal, suggesting that mitochondrial function is required only at levels of O 2 that support full development of ⁇ ⁇ m and OxPhos.
  • the mitochondrial agonist has no effects at 20% or 0.5% O 2 . At 20% O 2 , this suggests that differentiation is already maximized 7 days after FGF4 removal to the point that ATP is depleted and rate-limiting. At 0.5% O 2 , this suggests there are no agonist's effects because mitochondria are inactive due to high PKM2 diverting carbon from the mitochondria, and insufficient COX and O 2 to enable OxPhos. Taken together, these lines of evidence suggest that full mitochondrial activity is needed to enable full differentiation after the mitochondria are fully active three days after FGF4 removal.
  • HIFs induced hypoxia inducible factors
  • ROS reactive oxygen species
  • SAPK knockouts do not affect placentation in a normal vivarium (Xie, 2011; Rappolee, Reproduction, 145:R139, 2013 (Rappolee, 2013) so it will be important to study the effects of gestational hypoxic stress in SAPK knockouts.
  • Gestational hypoxia has revealed that placental and uterine hormones that are unnecessary during normal development become essential during hypoxic stress in vivo. Xie, 2011; Rappolee, 2013. In addition, gestational hypoxia produces a phenotype in vivo that has similarities with the phenotype observed here during hypoxia in culture. In both cases there was an increase in pTGC and a suppression of labyrinthine placenta.
  • TF Transcription factor
  • Fauque PLoS One, 5:e9218, 2010 ; Zhong, 2010. Perturbations of the embryo during the critical period of implantation frequently lead to loss of the pregnancy. Smart, Clin. Reprod. Fertil., 1:177, 1982 ; Wilcox, N. Engl. J. Med., 319:189, 1988 . Understanding the integration of stress enzyme signaling of the developing embryo may help to improve early pregnancy success rates, and avoid or mitigate long-term negative effects on the health of offspring.
  • TGCs trophoblast giant cells
  • TSCs placental trophoblast stem cells
  • Murine embryonic stem cells derived from the inner cell mass (ICM) of an E3.5 blastocyst are also highly sensitive to extrinsic signaling.
  • Extracellular signal regulated kinase (ERK) signaling can induce differentiation of mESCs; its suppression allows pluripotent stem cells to be derived from refractory mouse strains, and also allows the self-renewal of mESCs in culture. Burdon, Cells Tissues Organs, 165:131, 1999 .
  • Phosphoinositide 3-kinase PI3K regulates both the proliferation and pluripotency of mESCs, in part by its ability to maintain Nanog expression. Storm, J.
  • Pluripotency in both murine embryonic stem cells (mESCs) and human embryonic stem cells (hESCs) is maintained by a network of TFs-Oct4, Sox2, and Nanog-which suppress the differentiated state. Boyer, Cell, 122:947, 2005 ; Lee, Cell, 125:301, 2006 .
  • the TF Rex1 is another common marker of the pluripotent state.
  • Toxicological stressors can decrease potency in hESCs via a decrease in Oct4, Sox2, and Rex1 that potentially leads to abnormal differentiation. Pal, J. Cell. Physiol., 226:1583, 2011 ).
  • Oct4 maintains pluripotency in part by suppressing trophectoderm in both the ICM of the embryo and in the derivative mESCs in culture. Nichols, Cell, 95:379, 1998 ; Niwa, Nat. Genet., 24:372, 2000 (Niwa, 2000). A loss of 50% of Oct4 levels results in differentiation to trophectoderm, while a 50% increase above normal expression triggers differentiation to the early appearing primitive endoderm (PrEndo). Niwa, 2000. This is a reflection of the transient higher levels of Oct4 in the delaminating primitive endoderm derived from ICM of the E3.5 blastocyst. Palmieri, Dev. Biol., 166:259, 1994 .
  • Oct4 is required for differentiation of extraembryonic endoderm (ExEndo) by non-cell autonomous fibroblast growth factor (FGF)4 function and by cell autonomous upregulation of ExEndo TFs.
  • FGF fibroblast growth factor
  • Nanog suppresses PrEndo and its derivative ExEndo expression in the blastocyst. High Nanog expression is found only in pluripotent cells; low expression sensitizes mESCs to differentiation signals, committing them to PrEndo and later ExEndo lineages. Chambers, Nature, 450:1230, 2007 ; Singh, Stem Cells, 25:2534, 2007 . Rex1 expression correlates strongly with pluripotency in mESCs (Sharova, supra); its expression is lost as mESCs differentiate to either PrEndo/ExEndo or the later-appearing embryonic ectoderm (EmEcto). Rathjen, J. Cell.
  • Rex1-homozygous-null-mutant mESCs can be isolated, but have a higher spontaneous differentiation rate to all lineages. Scotland, Dev. Dyn., 238:1863, 2009 .
  • Rex1-null embryos express some visceral endoderm markers at lower levels ( Masui, BMC Dev. Biol., 8:45, 2008 (Masui, 2008)), suggesting a role for Rex1 in ExEndo differentiation.
  • cells of the ICM and its immediate successor lineages, PrEndo and EmEcto may be identified by measuring the relative quantitative expression of potency TFs.
  • the current Example used hyperosmotic stress to concurrently activate multiple stress enzyme signaling pathways in mESCs.
  • the stress-enzyme-dependent changes in TFs Oct4, Nanog, Sox2, and Rex1 were subsequently measured to reveal the impact of stress on pluripotency.
  • 5 protein kinases were revealed to affect the kinetics and/or the allocation of lineages from differentiating mESCs.
  • hyperosmotic stress did not trigger morphological differentiation of mESCs cultured in monolayer, it primed mESCs to initiate 1 st -lineage PrEndo/ExEndo differentiation, altering the response of mESCs to differentiation cues.
  • Reagents.MG132, lactacystin, and sorbitol were from Sigma (St. Louis, MO).
  • Enzyme inhibitors LY294002, U0126, PD98059, SB202190, AKTi, and L-JNKi-1 were from Calbiochem (La Jolla, CA).
  • Amido black was from MP Biomedicals (Solon, OH).
  • Rabbit polyclonal to mouse Oct4 (sc-5279), goat anti-human Sox2 (sc-17320), anti-rabbit MEK1 (sc-219), and anti-mouse MEK2 (sc-13159) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
  • Antirabbit Nanog was from Chemicon/Millipore (AB5731; Billerica, MA) and Rex1 antibodies were from Abcam (AB28141; Cambridge, MA).
  • RNA primers (Oct4,Nanog, Rex1, Dab2, Lrp2, and Fgf5) were from Integrated DNA Technologies (Coralville, IA).
  • mESC-D3 cells ATCC, Manassas, VA were cultured in the absence of feeder cells in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) supplemented with 15% mESC-screened fetal bovine serum (HyClone, Logan, UT), 2mM L-glutamine, 1mM sodium pyruvate, 1mM nonessential amino acids, 0.1mM 2-mercaptoethanol (Sigma), and 1,000 U/mL murine leukemia inhibitory factor (LIF; Millipore, Temecula, CA) on 0.1% gelatin-coated dishes at 37°C in humidified air with 5% CO 2 . Masui, 2008.
  • Dulbecco's modified Eagle's medium Gibco, Grand Island, NY
  • mESC-screened fetal bovine serum HyClone, Logan, UT
  • 2mM L-glutamine 1mM sodium pyruvate
  • 1mM nonessential amino acids 1mM no
  • mESCs were cultured overnight after passaging before stimulation with sorbitol.
  • Osmolality was determined by freezing point depression using Advanced Instruments Wide Range Osmometer 3W2 (Advanced Instruments, Norwood, MA).
  • 3W2 Advanced Instruments Wide Range Osmometer
  • One liter of a very dilute aqueous solution at room temperature very nearly has a mass of 1 kg, so at low solute concentrations ( ⁇ 0.50 M), osmolarity and osmolality are considered equivalent.
  • Enzyme inhibition Enzyme inhibitors were chosen according to the specificity reported in the kinase inhibitor literature. Alessi, J. Biol. Chem., 270:27489, 1995 ; Davies, Biochem. J., 351:95, 2000 . Inhibitor concentrations were determined following dose response experiments for each inhibitor based on the range of concentrations determined from the mESC and kinase inhibitor literature. Alessi, supra; Davies, supra; Gross, Mol. Reprod. Dev., 70:324, 2005 ); Hamazaki, Mol. Cell. Biol., 26:7539, 2006 ; Bain, Biochem. J., 408:297, 2007 ; Lee, Cell Prolif., 41:230, 2008 .
  • Cell accumulation and apoptosis were assayed by counting cells using a hemocytometer following trypan blue exclusion. MESCs were trypsinized, plated, and cultured overnight to allow for adaptation after passage. Time-zero counts were taken at least 1 day after passage and all subsequent counts were normalized to this. Apoptosis was measured by immunoblot for cleaved caspase 3. Mullen and Critser, Methods Mol. Biol., 254:393, 2004 .
  • Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE Healthcare, Waukesha, Wl), blot was scanned to obtain an electronic image, and intensity of protein bands was quantified using Image J analysis software (rsbweb.nih.gov) and normalized to amido black staining. Aldridge, J. Neurosci. Methods, 172:250, 2008 . Data are expressed as the change in expression relative to no treatment at time zero.
  • RT-qPCR primer sequences One microliter of cDNA template was added to 1 mL of both the forward and reverse primers for each specific transcript and 10 mL of Fast SYBR Green Master Mix (Applied Biosystems) for the RT-qPCR. Primer sequences are shown in Table 5. Table 5. RT-qPCR primer sequences.
  • Primer pairs were checked for specificity using BLAST analysis and were checked by both agarose gel electrophoresis and thermal dissociation curves to ensure amplification of a single product, and to rule out formation of primer dimers during the RT-qPCR.
  • the RT qPCR cycling parameters were as follows: enzyme activation, 95°C for 20 s; denature, 95°C for 3 s; and anneal/extend, 60°C for 30 s for a total of 40 cycles.
  • the expression of the target genes was quantified against that of two internal reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18s ribosomal RNA subunit (18s rRNA).
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • 18s ribosomal RNA subunit 18s rRNA
  • GAPDH was determined to be the most stable of 10 reference genes tested during mESC differentiation ( Murphy and Polak, Tissue Eng., 8:551, 2002 ; Willems, Int. J. Dev. Biol., 50:627, 2006 ), and 18s rRNA was selected due to its stability during hyperosmolar conditions in TSCs. Liu, supra. Fold change was determined using the ddCt method. Livak and Schmittgen, Methods, 25:402, 2001 . Data are expressed as the fold change in expression relative to no treatment at time zero.
  • Osmolality was measured at 330 ⁇ 4 mOsm/kg H 2 O before sorbitol addition (described in "Materials and Methods" section; Zhong, 2007), and 531 ⁇ 7 mOsm/kg H 2 O after addition of 200 mM sorbitol.
  • osmolality and osmolarity can be considered equivalent because the mass of 1 L of a dilute aqueous solution is very close to 1 kg.
  • the osmolarity of mouse uterine fluid has been reported as 330 mOsm. Harris, Theriogenology, 64:992, 2005 . Thus the described baseline media were isotonic with uterine fluid.
  • mESCs were cultured for 72 hr in three experimental conditions: isosmotic media +LIF, isosmotic media -LIF, and hyperosmotic media +LIF.
  • Cell counts were done ( FIG. 13B ) and micrographs were taken at 24, 48, and 72 hr of culture ( FIG. 13D ). Stress slowed growth rates from an overall doubling rate of 17.4 hr in untreated cells +LIF to 30.8 hr in treated mESCs ( FIG. 13C ).
  • apoptosis was assayed for by probing for the small cleavage product generated when caspase 3 is activated. It has been reported that mESCs do not express a functional death ligand (Fas/FasL) system. Brunlid, Stem Cells, 25:2551, 2007 . Nevertheless, caspase 3 is activated in both the extrinsic (i.e., extracellular induction) and intrinsic (mitochondrial) apoptotic pathways. This cleavage product was detected following 1-2 hr of stress (14% of cells; FIG.
  • Hyperosmotic activation of signaling enzymes A small subset of the 500 PKs in the kinome typically respond to stress stimuli, suggesting these as candidates for mediating cellular responses to hyperosmotic stress. Zhong, 2010; Xie, 2011. After screening 21 signaling kinases in 11 subfamilies using 14 inhibitors, 5 kinases in the mitogen activated protein kinase (MAPK) and PI3K families were identified as producing differentiation-priming effects in hyperosmotic conditions. To determine the kinetics of activation of these five kinases in the described system, mESCs were treated with 200 mM sorbitol for 1 hr.
  • MAPK mitogen activated protein kinase
  • activated (i.e., phosphorylated) enzymes were assayed by western blot analysis ( FIG. 14 ), including phospho-p38MAPK, phospho-JNK, phospho-MEK1/ 2, and phospho-AKT (the downstream effector of PI3K signaling).
  • Activated p38MAPK was 1 st detected at 10 min of stimulation; activation persisted throughout 1 hr of stimulation.
  • activated JNK was 1 st detected at 20 min of stimulation, and persisted throughout 1 hr of stimulation.
  • Activated MEK1/2 and activated AKT were present endogenously at low levels at time zero; sorbitol stimulated higher activation levels of each.
  • Increased MEK1/2 activation was stimulated within the 1 st 10 min of stress exposure, but returned to baseline levels by 20 min. In contrast, increased AKT activation occurred by 20-30 min of stimulation, and persisted through 1 hr of sustained stress exposure.
  • Efficacy of enzyme inhibitors was tested under stress conditions ( FIG. 15 ). LY294002 and AKTi inhibited AKT activation by an average of 88% and 95%, respectively.
  • PD98059 directly inhibits new MEK1 activation; its efficacy was determined by measuring the activation of MEK1's downstream targets, ERK1/2.
  • PD98059 inhibited ERK1/2 activation by an average of 30%. This is consistent with the literature that describes the mechanism of action of PD98059; it binds to inactive MEK1 thus preventing new activation, but does not shut off endogenously active MEK1. Dudley, Proc. Natl. Acad. Sci.
  • FIG. 16A illustrates the relative expression of these TFs in the undifferentiated ICM, modeled by mESCs, and each of the potential downstream lineages of mESCs, PrEndo that gives rise to other ExEndo lineages, and EmEcto.
  • Expression levels of Oct4, Sox2, Nanog, and Rex1 were determined at multiple time points up to 24 hr by western blot analysis and RT-qPCR.
  • Hyperosmotic stress rapidly activated the differentiation program, with expression of Oct4, Nanog, Rex1, and Sox2 TF proteins decreasing within the 1 st hour and reaching a nadir between 2 and 4 hr of continued stress ( FIG. 16B ).
  • the nadir of Nanog expression was more variable than the other three TFs, occurring as early as 2 hr in some experiments or as late as 6 hr of stress.
  • the overt differentiation program was aborted, and the decline in Oct4, Sox2, and Nanog expression halted.
  • expression of these three TF proteins rebounded toward the unstressed baseline with both Sox2 and Nanog achieving complete, robust recovery to their prestressed protein levels ( FIG. 16B ).
  • Nanog, Oct4, and Rex1 expression was adequate to identify mESC or subsequent lineages, Sox2 expression was not continued. Nanog and Oct4 recovery was maintained through 72 hr of ongoing stress ( FIG. 16E , Oct4, data not shown). Recovery at the mRNA level also occurred, with both Oct4 and Nanog moving toward their unstressed baseline, although this recovery had not reached significance at 24 hr ( FIG. 16C ).
  • Rex1 protein levels did not rebound, remaining at ⁇ 40% of its unstressed levels throughout the 24-h time course ( FIG. 16B ).
  • Rex1 mRNA levels however, recovered to 70% of their unstressed levels by 24 hr ( FIG. 16C ).
  • Stress induced a rapid, transient loss of protein and mRNA in potency TFs, but this loss was reversed in some TFs as mESCs adapted to the stress.
  • Rex1 protein was exceptional in its persistent stress-induced suppression through 24 hr.
  • Sorbitol induced significantly higher levels of Lrp2 and Dab2 expression compared with either of the time-matched controls ( FIG. 16D ).
  • 24 hr of LIF removal was sufficient for unstressed cells to upregulate the EmEcto marker Fgf5
  • the presence of sorbitol suppressed this upregulation in stressed cells.
  • the stress-induced loss of Rex1 correlated with upregulation of markers of the early appearing PrEndo/ExEndo lineages, but suppression of the later EmEcto lineage marker.
  • mESCs were treated with one of two proteasome inhibitors, MG132 ( FIG. 17 ) and lactacystin ( FIG. 18A-18D ). Both inhibitors prevented the stress-induced loss of Oct4, Nanog, and Rex1. Oct4 loss was reversed by 75%, Nanog by 93%, and Rex1 by 310% in 4 hr of proteasome inhibition by MG132.
  • MEK1 and other enzymes trigger mESC initial stress response: initiation of differentiation program.
  • mESCs were cultured in the presence of pharmacological inhibitors and hyperosmotic stress for 4 hr to identify the enzymes that mediated the stress-induced loss of potency TF proteins, reported in FIG. 16B .
  • the complete results of p38, JNK, MEK1/2, and PI3K inhibition during 4 hr of hyperosmotic stress in mESCs are summarized in Table 6. Table 6.
  • Table 6 TF expression levels during 4 hr of hyperosmotic stress +/- enzyme inhibition in mESC monolayer culture.
  • MEK1 activation triggered a loss of expression of all three TFs; when its stress-induced activation was prevented by PD98059, expression of both Oct4 and Rex1 remained at the unstressed baseline, while Nanog expression increased to 2.5 times its unstressed level ( FIG. 19A ). This effect was repeated for Rex1 during inhibition with the MEK1/2 inhibitor U0126 ( FIG. 19B ); due to inhibition of both MEK1 and MEK2 during stress exposure, Rex1 expression remained at unstressed levels. This suggests that MEK1 was the regulator of Rex1 protein destruction during stress.
  • p38MAPK and other signaling kinases produced mESC adaptive response to hyperosmotic stress: differentiation program aborted, and pluripotency restored at 24 hr.
  • the enzymatic mechanisms that mediated the recovery of potency TF proteins during stress that persisted for 24 hr was next tested.
  • mESCs were cultured in the presence of hyperosmotic stress for 24 hr with or without pharmacological inhibitors of PKs; the complete results of p38MAPK, JNK, MEK1/2, and PI3K inhibition during 24 hr of hyperosmotic stress are summarized in Table 4.
  • p38MAPK signaling mediated the recovery of Oct4 and Nanog proteins to their unstressed baselines during 24 hr of stress ( FIG.
  • JNK-dependent signaling produced persistent suppression of Rex1 during 24 hr of hyperosmotic stress.
  • p38MAPK signaling was not able to reverse Rex1 suppression, neither was PI3K signaling ( FIG. 21A ).
  • JNK signaling suppressed Rex1 expression during 24 hr of hyperosmotic stress ( FIG. 21A ).
  • mESCs were subjected to only 4 hr of hyperosmotic stress and then returned to iso-osmotic media.
  • Rex1 expression at 24 hr recovered to baseline; this indicates that continued stress was required for persistent Rex1 suppression ( FIG. 20B ).
  • the persistent suppression of Rex1 throughout the duration of stress stimulation suggests that at least a subpopulation of mESCs may be primed by stress for differentiation by maintaining chronic Rex1 loss as well as transient loss of Oct4, Nanog, and Sox2.
  • MEK1 mediated potency loss at the 4-h nadir of Oct4 and played a role in regain of Oct4, Nanog, and Rex1 from 4 to 24 hr.
  • the mediator of regain of Oct4 by MEK1/2 is not clear.
  • MEK1 is a weaker enzyme that blocks the stronger enzyme MEK2 in a heterodimer ( Catalanotti, Nat. Struct. Mol. Biol., 16:294, 2009 ), and both MEK1 and MEK2 are expressed in TSCs/ESCs/blastocysts (Wang, supra) and should heterodimerize.
  • PD98059 suppresses MEK1 and reactivates MEK2 that reverses stress-induced Oct4 loss from 0 to 4 hr.
  • U0126 inhibits both but importantly MEK2, blocking Oct4 recovery from 4 to 24 hr.
  • MEK2 protects Oct4 from 0 to 4 hr unless MEK1 activity is dominant and reverses Oct4 loss from 4 to 24 hr if MEK2 activity becomes dominant (Table 7).
  • Activity and chemical inhibitor data for MEK1 and MEK2 support this but further attempts to test this hypothesis using siRNA to MEK1 and MEK2 failed due to insufficient specificity of the knockdowns (data not shown).
  • JNK mediates both Nanog loss from 0 to 4 hr and continuing Rex1 loss between 4 and 24 hr. Both JNK-dependent effects would mediate predisposition toward endoderm, which is suppressed by Nanog (Frankenberg, supra; Hamazaki, J. Cell. Sci., 117:5681, 2004 ; Mitsui, Cell, 113:631, 2003 ), although Rex1 may mediate later ectoderm and specific subtypes of extraembryonic endoderm. Scotland supra; Masui, 2008.
  • JNK prioritizes differentiation of TSCs stressed by hyperosmotic sorbitol, benzopyrene, or hypoxic O 2 below 2%, by inducing 1 st lineage (Xie, 2014; Awonuga, 2011; Abdallah, Fertil. Steril., 92(3):S136, 2009 ) and suppressing later lineages. Xie, 2014. JNK appears to mediate stress-induced endoderm, the 1 st lineage arising from ESCs.
  • Lrp2 marks the earliest allocation of primitive endoderm in the E3.5 blastocyst and is accompanied by a suite of other proteins, such as cubilin and megalin (e.g., SEQ ID NO:69), that are also expressed in the earliest ExEndo in the blastocyst and are part of lipid uptake mechanisms shared by many absorptive epithelia. Frum, supra; Maurer & Cooper, J. Cell. Sci., 118:5345, 2005 ; Assemat, Biol.
  • cubilin and megalin e.g., SEQ ID NO:69
  • Dab2 arises during primitive endoderm lineage fixation at E4.5 and anchors the absorptive complex in the apical surface where it may function to feed the endoderm and adjacent ICM in the blastocyst or inner stem cells subjacent to the outer visceral endoderm of embryoid bodies. Frum, supra; Yang, J. Biol. Chem. 282:13114, 2007 .
  • Hyperosmotic stress begins to induce this absorptive capability in mESCs in monolayers, similar to the induction by several stressors of the placental hormone PL1 from TSCs (Xie, 2014; Awonuga, 2011; Liu, supra; Rappolee, 2013) that increases nutritional supply to the fetal-maternal interface in vivo.
  • PI3K and p38MAPK protect potency after adaptation to stress, leading to recovery of Nanog/Rex1 and Nanog/Oct4 between 4 and 24 h, respectively (Table 6).
  • Oct4 recovery may occur for several reasons. One is that a sufficient, successful stress response occurs during the 1 st 4 hr and allows substantial ESC proliferation and accumulation, and thus induction of full differentiation is not required to compensate for fewer cells. Alternately, the function of early stem cells is to retain potency and divide exponentially between days E4.0 and E11.0 ( Embryogenesis in mammals. (1976). Elsevier, Amsterdam; New York ; McLaren. Embryo growth during the immediate postimplantation period. In Embryogenesis in mammals. (1976).
  • Mao found that mESCs maintained a pluripotent phenotype during long-term exposure to hyperosmolarity, although proteins involved in both protein synthesis and degradation via the ubiquitin-proteasome system were decreased.
  • Mao J. Proteome Res., 7:3968, 2008 .
  • hyperosmotic stress at these levels causes an 80% decrease in new translation within the 1 st 30 min. Patel Eur. J. Biochem., 269:3076, 2002 .
  • stress decreases new translation and increases potency factor degradation, rapid, adaptive programmatic changes are possible.
  • the ubiquitin-proteasome system has been reported to have a role in the self-renewal and differentiation of both human and mouse ESCs (reviewed in Naujokat & Saric, Stem Cells, 25:2408, 2007 ). Inhibition of proteasomes decreased levels of four: Oct4, Sox2, Nanog, and Rex1 mRNAs ( Vilchez, Nature, 489:304, 2012 ) and Oct4 and Nanog mRNAs in unstressed human ESCs and thus proteasome function is proposed to be part of potency maintenance. It is not clear from these studies or the Examples described here whether stress would increase proteasome-dependent protein destruction in human ESCs or proteasome-dependent increase in potency mRNA occurs in unstressed mouse ESCs, respectively.
  • the activity of the 20S subunit of the proteasome is normally upregulated during mESC differentiation ( Hernebring, Proc. Natl. Acad. Sci. USA, 103:7700, 2006 ); its suppression during hyperosmotic stress supports the finding that mESCs are not enabled to differentiate fully during extended hyperosmotic stress.
  • Oct4 protein ranges from 1.5 to 8 hr. A half-life of 90 min has been reported for Oct4 protein in undifferentiated P19 ( Sax, PLoS One, 4:e4467, 2009 ), 6-8 hr in NIH3T3-overexpressing Oct4 protein ( Wei, J. Biol. Chem., 282:21551, 2007 ), 6.9 hr for Oct4 mRNA in mouse ESCs (Sharova, supra), and a few hours for Oct4 mRNA in differentiated cells undergoing reprogramming. Taranger, Mol. Biol. Cell, 16:5719, 2005.
  • Oct4 is required to exclude Cdx2 in nonpolar inner cells of the blastocyst ( Niwa, Cell, 123:917, 2005 ) and is required for FGF4 secretion that sustains adjacent trophectoderm ( Arcuri, Mol. Cell. Endocrinol., 141:13, 1998 ; Ma, Dev. Biol., 154:45, 1992 ) and primitive endoderm cells in the three-dimensional (3D) blastocyst. Frum, supra; Frankenberg, supra; Cho, Development, 139:2866, 2012 ; Krawchuk, Dev. Biol., 384:65-71, 2013 .
  • Oct4 also plays a transient, cell-autonomous role in establishing primitive endoderm.
  • Oct4 is a stress response factor that controls stem cell metabolism in mESCs in culture before and during stress ( Kang, Trends Biochem. Sci., 34:491, 2009 ; Kang, Genes Dev., 23:208, 2009 ) and metabolism of cultured mouse embryos. Frum, supra.
  • the cohort of potency TFs with Oct4 at its apex is dynamically poised under normal circumstances to regulate potency, allocation, stem cell metabolism, and stress responses.
  • Nanog has a half-life of 2 hr in human ESCs, and is controlled by proteasomal regulation via the PEST motif.
  • Ramakrishna Stem Cells Dev., 20:1511, 2011 .
  • mESCs a half-life of 5.2 hr has been reported for Nanog mRNA.
  • Rex1 half-life has been reported to be from 30 min ( Gontan, Nature, 485:386, 2012 ) to 2.2 hr (Sharova, supra) in mESCs (Gontan, supra).
  • inhibition of the proteasome for 4 hr led to a 3-fold increase in Rex1 expression, suggesting a short half-life of this protein.
  • Rex1 was the only pluripotency marker that did not recover to normal expression levels during stress is unique, as it normally recovers quickly during fluctuations in expression. Toyooka, supra. This may be due to increased protein destruction, JNK-dependent silencing of the Rex1 promoter despite Oct4 (Boyer, supra), or JNK-dependent phosphorylation of Oct4 that decreases binding to the Rex1 promoter.
  • Rex1-negative mESCs were defective in some visceral endoderm markers although the major marker, ⁇ -feto protein (AFP), was expressed (Masui, 2008) and Rex1-/-F9 teratocarcinoma stem cells were only able to differentiate to parietal endoderm. Thompson & Gudas, Mol. Cell. Endocrinol., 195:119, 2002 .
  • the data suggest that Rex1 plays a role in directing lineages of endoderm differentiation in F9 cells but data should be interpreted cautiously as, unlike mESCs, AFP requires Rex1 expression and F9 cells may not emulate complex regulatory mechanisms of mESCs.
  • JNK activation by continuous hyperosmotic stress suppressed Rex1 expression was not adequate to trigger outright differentiation of mESCs. JNK may not be activated long enough to irreversibly commit ESCs to differentiation, or there may be insufficient activation of other enzymes needed to complement JNK-induced differentiation, or insufficient 3D interactions may occur. Unlike TSCs, ESCs may require the 3D interactions available in embryoid bodies. However, it was not defined whether JNK directly decreased Rex1 protein or indirectly suppressed it during induction of differentiation. In each case of stress-induced differentiation of mESCs and TSCs, JNK was active within a pathway required to produce a new lineage, but in none of the cases was it capable of initiating differentiation on its own.
  • JNK may work with other differentiation cues and enzymatic mechanisms. But during the stress response, the activation of multiple pathways with competing effects prevented outright or complete differentiation of large cell subpopulations. For example, JNK suppressed Rex1 expression during 24 hr of stress, while PI3K signaling was simultaneously maintaining Rex1, preventing total Rex1 protein loss. This additional signaling masked but did not negate JNK's action on Rex1. Of course if a single ESC lost the three potency TFs undergoing transient stress-induced loss, and the continuing loss of Rex1 for a long enough period, then it might differentiate irreversibly.
  • BMPs are a serum component that induces expression of inhibitor of differentiation genes that block expression-lineage-specific TF function and facilitate the self-renewal response to LIF/STAT3. Ying, Cell, 115:281, 2003 . In the described system, the integration of all these signals during hyperosmotic stress led to preservation of a pluripotent population; stress was not dominant in overt changes in mESC monolayers. However, in vivo the pluripotent ICM is a transient stage that stem cells move through to populate the lineages that will eventually make up the embryo and its support cells.
  • LIF in the four-cell-stage embryo and interleukin-6 at the blastocyst stage are necessary to maintain phosphorylated STAT3 and Oct4 in order to maintain pluripotency in vivo.
  • Do, Genes Dev., 27:1378, 2013 If stress occurred in an environment that was more characteristic of the nonepithelial ICM, one where differentiation was not repressed, then the stress activation of the differentiation program may proceed unimpeded. Thus, stress may drive preimplantation ICM to Ex-Endo but suppress other lineages that arise at gastrulation.
  • Example 3 The embryo undergoes stress in vivo and in vitro and this decreases developmental rates, stem cell growth rates and causes potency loss ( Puscheck, Molecular Biology of the Stress Response in the Early Embryo and its Stem Cells. In: Cell Signalling During Mammalian Early Embryo Development. Henry Leese DB ed. Springer, 2015 (Puscheck, 2015, MB); Xie, 2011).
  • ESCs respond to hyperosmotic stress with transient loss of Oct4 and long-term loss of Rex1 proteins in a proteasome-dependent manner and this correlates with induction of 1 st lineage and suppression of later lineages during stress (Example 2; Slater, Stem Cells Dev., 23:3049, 2014 ).
  • Early embryos at the 2-cell and blastocyst-stage also undergo stress-induced potency loss as do placental trophoblast stem cells (TSCs) derived from the blastocyst (Xie, 2013; Zhou
  • Oct4 and Rex1 are DNA-binding transcription factor that mediates stemness in gametes and early embryos and in pluripotent cells through the start of gastrulation ( Pesce & Scholer, Mol. Reprod. Dev., 55:452, 2000 ).
  • Oct4 null lethality occurs at the blastocyst stage when ICM lose pluripotency and fail to synthesize FGF4 ( Nichols, Cell, 95:379, 1998 ) that maintains adjacent polar trophectoderm (Chai, 1998).
  • Oct4 promoter methylation is diminished in oocytes undergoing in vitro maturation (IVM) ( Milroy, Fertil.
  • Oct4 expression is decreased in embryos derived from smoke-exposed mouse females ( Huang, Fertil. Steril., 92:1456, 2009 ).
  • Oct1 and Oct4 have been used to test for toxic stress in HTS of embryonic stem cells ESCs ( Pal, J. Cell. Physiol., 226:1583, 2011 ) and both transcription factors have stress domains that prepare stem cells for stress and are phosphorylated and regulate the stress response ( Kang, Genes Dev., 23:208, 2009 ).
  • Rex1 is also a transcriptional factor that is lost from ICM as they differentiate to either extra-embryonic primitive endoderm or embryonic primitive ectoderm fates (Toyooka, supra) but its null is not lethal at the blastocyst stage and it is not required to initiate and maintain pluripotency of ESCs ( Rezende, Dev. Biol., 356:370, 2011 ).
  • Rex1-driven reporters should be more useful than Oct4-driven reporters to create HTSs for toxicants or other stressful stimuli that negatively affect embryo and stem cell stemness and differentiation, thus depleting stem cells and imbalancing differentiation.
  • lentivirus infection can be used ( Kaufman, Theriogenology, 82:1043, 2014 ) and in place of antibiotic selection ESCs are passaged in the normal manner but fluorescence activated cell sorted (FACS) to select stem cells reporting pluripotency. This creates viable potency reporter ESC lines with normal growth rates and without cell loss during infection and selection. These mESCs also report potency factor activity loss using 1) visual microscopic inspection, 2) microplate readers, 3) FACS, and 4) immunoblots. The assays provide similar results as in Example 2.
  • the analysis by flow cytometry also provides important new information on the size of the bright (potent) subpopulation that goes to zero potency and the microplate gives a platform of high throughput analysis of toxicants or other potential stressors that may induce potency loss and for performing high throughput screens to further elucidate the mechanisms of potency loss.
  • Germline competent mESC-D3 cells were purchased from ATCC (Manassas, VA).
  • DMEM medium was from HyClone (Logan, UT).
  • Gibco TM glutamine and sodium pyruvate supplement solutions were from Life Technologies (Grand Island, NY).
  • ESC-qualified EmbryoMax fetal bovine serum, 0.1% gelatin solution and ESGRO TM Mouse LIF medium supplement were from EMD Millipore (Billerica, MA).
  • Oct4 promoter reporter lentiviral particles that express the green fluorescent protein copGFP and TranDux TM transduction reagent were from System Biosciences (Mountain View, CA).
  • Rex1 promoter reporter lentiviral particles that express the red fluorescent protein mApple were from Allele Biotechnology (San Diego, CA).
  • Rabbit anti-Rex1 antibody was from Abcam (Cambridge, MA).
  • Rabbit anti-cleaved caspase 3 antibody, Rabbit anti-p-actin antibody and HRP-conjugated second antibodies were from Cell Signaling Technology (Danvers, MA).
  • Pierce RIPA lysis buffer, protease inhibitor cocktail and BCA protein assay reagents were from Thermo Scientific (Rockford, IL).
  • ECL chemiluminescence reagent was from GE Healthcare Bio-Sciences (Pittsburgh, PA).
  • MEM nonessential amino acid solution, sorbitol, 2-mercaptoethanlol and other chemicals were from Sigma (St. Louis, MO).
  • mESCs Low stress production of single and double viable potency activity reporter mouse ESCs.
  • mESCs were cultured in 24-well plates pre-coated with 0.1% gelatin. The starting confluence of cells was 20% or 100,000 cells/well. The cells were incubated in 37°C for 2-3 hr for attachment and readiness for virus infection.
  • Virus infection medium was made by mixing Oct4 and/or Rex1 promoter reporter lentiviral particles into regular growth medium supplemented with TransDux. The final concentration of the viral particles was 10 6 /ml. The infection medium was applied to mESCs at 400 ⁇ l per well. After infection, the cells were cultured for 3 days to allow the expression of the fluorescent reporter proteins.
  • the infected cells were subjected to flow cytometry sorting using BD FACS Vantage SE cell sorter (BD Biosciences, San Jose, CA) at the Wayne State University Microscopy, Imaging and Cytometry Resources (MICR) core facility. Pure, fluorescent cells were obtained after two FACS repeats. But, as indicated in FIG. 25A and 25B and as previously reported by Toyooka and colleagues Toyooka, supra) heterogeneity reestablishes after sorting and culture and normally a small population becomes Rex1 dim.
  • BD FACS Vantage SE cell sorter BD Biosciences, San Jose, CA
  • MICR Imaging and Cytometry Resources
  • Germline competent mESC-D3 cells ATCC, Manassas, VA, ( Doetschman, J. of Embryology and Experimental Morphology, 87:27, 1985 ) were cultured in the absence of feeder cells in DMEM (Gibco, Grand Island, NY) supplemented with 15% mESC-screened fetal bovine serum (HyClone, Logan, UT), 2mM L-glutamine, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol (Sigma, St.
  • mESCs were cultured overnight after passaging before stimulation with sorbitol. Osmolality of ESC media with and without added 200 mM sorbitol was determined as in Example 2.
  • mESCs that stably express Oct4-copGFP and/or Rex1-mApple promoter reporters were plated on 96-well plates at a starting confluence of 5%. Four identical plates were made for the time course study. Cells were grown for 24 hr in incubator till 10% confluence was reached; an addition of stimuli was designated time zero. The cells were stressed for a time period selected from 1, 2, or 3 days. At each time point, the medium was removed and the cells were rinsed with D-PBS. At 50 ⁇ l/well, ESCs were measured for fluorescence using the Synergy H1 microplate reader (BioTek, Winooski, VT).
  • the excitation and detection wavelengths for Oct4-copGFP were 485nm and 528nm, respectively.
  • the excitation and detection wavelengths for Rex1-mApple were 568nm and 611nm, respectively.
  • the cells were lysed with 50 ⁇ L of lysis buffer (1% NP-40, 0.5% TX-100, 100 mM NaCl, 2mM EDTA, 50 mM Tris-HCI, and pH 7.5) and measured for fluorescence with the same parameters above. After fluorescence determination, the cell lysates were further measured for total protein concentrations by adding 100 ⁇ l of Pierce BCA protein assay reagent (Thermo Scientific Inc.) following the manufacturer's instructions. Finally, the fluorescence readings from either live-cells or lysates were normalized to total protein concentration readings, in which higher readings represent higher pluripotency of the cells.
  • Flow cytometric analysis of mESCs expressing pluripotency reporters Fluorescence determination by the plate reader reported an averaged biochemical result from a population of cells inside the tissue culture vessel. In order to get insight into the heterogenic nature of the cells that respond to the same stress condition, a flow cytometry assay that provides quantification of cells that show different levels of pluripotency under stress was used.
  • mESCs expressing potency reporter Oct4-CopGFP or Rex1-mApple were plated at a starting confluence of 10% and grown for 24 hr until reaching 25% confluence. This time point was designated as time zero. The cells were then treated with different sorbitol concentrations for 1-3 days.
  • Non-stressed mESCs that express Oct4-copGFP and/or Rex1-mApple and the non-modified, parental mESCs were used to set up the parameters that define the fluorescent cells and non-fluorescent cells.
  • the right peak represents the bright fluorescent potent ESCs.
  • the left peak represents the dim fluorescent non-potent ESCs.
  • An arbitrary vertical line is drawn here at the 5 x 10 3 position on the PE-A axis to separate the low potency ESCs.
  • the peak areas were calculated using Image J by counting the area pixels.
  • ESCs were trypsinized as for a normal passage, and a fluorescence-activated cell sorter (FACS) was used to purify Rex1-RFP ( FIG. 22D ), Oct4-GFP ( FIG. 22E ), and dual Rex1-RFP/Oct4-GFP expressing ESCs ( FIG. 22F ).
  • FACS fluorescence-activated cell sorter
  • Oct4-GFP fluorescence intensity was similar without ( FIG. 22G ) or with ( FIG. 22J ) stress, but Rex1-RFP fluorescence was clearly higher without stress ( FIG. 22H ) than with stress ( FIG. 22K ).
  • FIG. 221 In corroboration,-d culture of Rex1-RFP/Oct4-GFP dual expressing cells were not affected by culture without stress (FIG. 221), but stress decreased Rex1-RFP, removing red from yellow and re-exposing green fluorescence from Oct4-GFP ( FIG. 22L ).
  • FIG. 22L the visual data suggest that Rex1-RFP activity reporter is decreased in viable ESCs after 3 days of hyperosmotic stress.
  • the amount of stress-induced loss of Rex1-RFP or Oct4-GFP was next quantitated using a microplate reader. It was found that stress mediated by 200 mM sorbitol, despite the presence of LIF, induces a significant loss of Rex1-RFP but not Oct4-GFP ( FIG. 23A,23B ). This is in general agreement with the loss of Rex1 protein but not Oct4 protein after 24 hr of stress. However, the loss of Rex1 protein is greater than the loss of Rex1 activity detected by the multimerized Rex1 response elements driving RFP fluorescence assay in the microplate reader.
  • Rex1-RFP ESCs were cultured for 3 days with 0-300 mM sorbitol and assayed by immunoblot for endogenous Rex1 levels normalized to ACTB loading controls and with time zero unstressed Rex1 set to 100 as a baseline level.
  • stress-induced decrease in Rex1 activity in FIG. 24 were 20% and 70% at 200 mM and 300 mM sorbitol, respectively endogenous Rex1 protein losses were 30% and 70% at 200 mM and 300 mM, respectively ( FIG. 27 ).
  • Rex1-RFP potency activity reporter ESCs show consistency between micrographic analysis, biochemical averages from immunoblots and microplate reader, and subpopulation studies using flow cytometry. Endogenous Rex1 protein levels are consistent with Rex1-RFP activity levels measured in the same cells under the same stress exposures.
  • Rex1-RFP is a sensitive stress reporter and will enable an HTS for developmental toxicology of manufactured compounds, environmental pollutants, new Pharma and cosmetics and fluid collected during IVF ART protocols. These include follicular, vaginal, uterine, amniotic fluids and spent media. Hyperosmotic stress, which is a "gold standard” for discovery and characterization of stress enzymes (Xie, 2011), was used in this Example. There is also an exemplar set of toxicants used for the only government-validated ESC toxicology assay ( Genschow, European Centre for the Validation of Alternative Methods. Altern Lab Anim, 30:151, 2002 , and these will be used to further test the validity of the Rex1-RFP HTS.
  • the stress dose-dependent cell number of low potency cells can be determined by multiplying the fractional subpopulation size of cells with potency loss, which increases with stress and the decreasing cell numbers with increasing stress. This was done in Table 8 and compared with a previously published Venn diagram for compensatory differentiation ( Puscheck, 2015, MB; Rappolee, 2013), modified to produce FIG. 28 .
  • the absolute size of the subpopulation of potency loss is remarkably the same.
  • the subpopulation number with potency loss decreases, probably due to stem cell morbidity at these higher hyperosmotic stress levels (Example 2).
  • a Pdgfra-GFP ESC line can be tested for stress effects on differentiation of the 1 st lineage ESC-derived extra-embryonic endoderm that this line reports ( Artus, Development, 137:3361, 2010 ). This will provide an even more direct test of stress-induced differentiation and the regulation and number of cells in the subpopulation undergoing stress dose-dependent potency loss and differentiation increase. Without being bound by theory, it is hypothesized that Pdgfra-GFP cells will derive primarily from the parental dim subpopulation of Rex1-RFP cells, but differentiated cells may arise from the intermediate dim or even bright groups of Rex1-RFP cells, but at lower frequencies.
  • modified stem cells e.g., Rex1-RFP and Pdgfra-GFP potency and 1 st differentiated lineage ESC
  • HTSs human stem cells
  • any embodiment can be used in combination with any of the following markers of interest for pregnancy outcome including Cofillin (NM_ 005507), DIAPH1 (NM_001079812, NM_005219), ECT2 (NM_018098), MYLC2/MYL5 (NM_ 002477), DGCR8 (NM_022720), Dicer/DICER1 (NM_030621, NM_177438), TARBP2 (NM_ 004178, NM_134323, NM_134324), CPEB1 (NM_001079533, NM_001079534, NM_ 001079535, NM_030594), Symplekin/SYMPK (NM_004819), YBX2 (NM_015982), ZAR1 (NM_ 175619), CTNNB1 (NM_001098209, NM_001098210, NM_001098210, NM_00190
  • each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component.
  • the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.”
  • the transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts.
  • the transitional phrase “consisting of' excludes any element, step, ingredient or component not specified.
  • the transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment.
  • a material effect would cause a statistically-significant reduction in the ability of a method to detect stress in a stem cell.
  • the term "about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ⁇ 20% of the stated value; ⁇ 19% of the stated value; ⁇ 18% of the stated value; ⁇ 17% of the stated value; ⁇ 16% of the stated value; ⁇ 15% of the stated value; ⁇ 14% of the stated value; ⁇ 13% of the stated value; ⁇ 12% of the stated value; ⁇ 11% of the stated value; ⁇ 10% of the stated value; ⁇ 9% of the stated value; ⁇ 8% of the stated value; ⁇ 7% of the stated value; ⁇ 6% of the stated value; ⁇ 5% of the stated value; ⁇ 4% of the stated value; ⁇ 3% of the stated value; ⁇ 2% of the stated value; or ⁇ 1% of the stated value.
  • CE of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Developmental Biology & Embryology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Reproductive Health (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Gynecology & Obstetrics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Claims (6)

  1. Screening-Verfahren zum Erkennen von Stress in Stammzellen, die einer zu screenenden Verbindung ausgesetzt sind, wobei das Verfahren Folgendes umfasst:
    Messen der Expression oder Aktivität von Oct4; Sox2; Nanog; Rex1; TEAD4; Errß; Lrp2; Dab2; Fgf5; Pdgfra; Sox17; Gata4/6; PAX8; NEFL; TSHR; Brachyury; Laminin; AFP; Nestin; Goosecoid; MEK1; MEK2; und/oder AKT in der Gegenwart von Fibroblasten-Wachstumsfaktor 4 (fibroblast growth factor- FGF4) für murine Trophoblasten-Stammzellen (trophoblast stem cells - TSCs) oder Leukämieinhibitionsfaktor (LIF) für murine embryonale Stammzellen (embryonic stem cells - ESCs);
    Vergleichen der Messung mit einem Referenzwert von Expression oder Aktivität, der aus früheren Messungen von einer Population von Stammzellen abgeleitet ist, die der zu screenenden Verbindung nicht ausgesetzt waren; und
    Bestimmen, dass die Stammzellen Stress erfahren haben, wenn die Messung gegenüber dem Referenzwert statistisch signifikant verringert oder erhöht ist,
    wobei das Verfahren Verbindungen auf ihre Stresswirkungen auf die Stammzellen screent, und
    wobei eine statistisch signifikante Verringerung von Oct4; Sox2; Nanog; Rex1; TEAD4; oder Errß eine Stresserfahrung anzeigt, oder
    eine statistisch signifikante Erhöhung von Lrp2; Dab2; Fgf5; Pdgfra; Sox17; Gata4/6; PAX8; NEFL; TSHR; Brachyury; Laminin; AFP; Nestin; Goosecoid; MEK1; MEK2; oder AKT eine Stresserfahrung anzeigt.
  2. Verfahren nach Anspruch 1, das das Messen der Expression oder Aktivität von Oct4 und/oder Rex1 umfasst, wobei eine statistisch signifikante Verringerung im Vergleich zu dem Referenzwert eine Stresserfahrung anzeigt.
  3. Verfahren nach Anspruch 1, das das Messen der Expression oder Aktivität von Lrp2, Dab2, Fgf5 und/oder Pdgfra umfasst, wobei eine statistisch signifikante Erhöhung im Vergleich zu dem Referenzwert eine Stresserfahrung anzeigt.
  4. Verfahren nach Anspruch 1, das das Messen der Expression oder Aktivität von Pdgfra, Sox17, Dab2, Lrp2 oder Gata6 umfasst, wobei eine statistisch signifikante Erhöhung im Vergleich zu dem Referenzwert eine Stresserfahrung anzeigt.
  5. Verfahren nach Anspruch 1, das das Messen der Expression oder Aktivität von Brachyury oder FGF5 umfasst, wobei eine statistisch signifikante Erhöhung im Vergleich zu dem Referenzwert eine Stresserfahrung anzeigt.
  6. Verfahren nach Anspruch 1, das das Messen der Expression oder Aktivität von Rex1 und Pdgfra umfasst, wobei eine statistisch signifikante Verringerung von Rex 1 im Vergleich zu dem Referenzwert eine Stresserfahrung anzeigt und eine statistisch signifikante Erhöhung von Pdgfra im Vergleich zu dem Referenzwert eine Stresserfahrung anzeigt.
EP15831338.7A 2014-08-12 2015-08-11 Systeme und verfahren zur erkennung von stress von stammzellen und verwendungen davon Active EP3194623B1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201462036549P 2014-08-12 2014-08-12
US201562159675P 2015-05-11 2015-05-11
PCT/US2015/044701 WO2016025510A1 (en) 2014-08-12 2015-08-11 Systems and methods to detect stem cell stress and uses thereof

Publications (3)

Publication Number Publication Date
EP3194623A1 EP3194623A1 (de) 2017-07-26
EP3194623A4 EP3194623A4 (de) 2018-03-14
EP3194623B1 true EP3194623B1 (de) 2022-01-05

Family

ID=55304558

Family Applications (1)

Application Number Title Priority Date Filing Date
EP15831338.7A Active EP3194623B1 (de) 2014-08-12 2015-08-11 Systeme und verfahren zur erkennung von stress von stammzellen und verwendungen davon

Country Status (3)

Country Link
US (2) US20180363070A9 (de)
EP (1) EP3194623B1 (de)
WO (1) WO2016025510A1 (de)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019060702A1 (en) * 2017-09-22 2019-03-28 Wayne State University SYSTEMS AND METHODS FOR STRESS DETECTION AT RISK OF FALSE LAYER
EP3706745A4 (de) * 2017-11-09 2022-07-20 Dana-Farber Cancer Institute, Inc. Verfahren zur verhinderung der teratogenität von imidähnlichen molekülen und degradern/protacs auf imidbasis
EP3999660A1 (de) * 2019-07-19 2022-05-25 Toxys B.V. Verfahren zur bestimmung der wirkung eines mittels auf die embryonale entwicklung von säugetieren
CN110747271B (zh) * 2019-12-02 2023-07-18 延边大学 关于pci术后血管损伤-再狭窄程度的标记物
WO2023205576A2 (en) * 2022-04-20 2023-10-26 Cook Medical Technologies Llc Dual genetic reporters in human pluripotent cells for use as alternative to mouse embryo assay

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011029798A1 (en) * 2009-09-09 2011-03-17 General Electric Company Composition and method for imaging stem cells

Family Cites Families (45)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5525464A (en) 1987-04-01 1996-06-11 Hyseq, Inc. Method of sequencing by hybridization of oligonucleotide probes
US5202231A (en) 1987-04-01 1993-04-13 Drmanac Radoje T Method of sequencing of genomes by hybridization of oligonucleotide probes
GB8810400D0 (en) 1988-05-03 1988-06-08 Southern E Analysing polynucleotide sequences
CA1341469C (en) 1988-08-04 2004-12-28 Robert Lindsay Williams In vitro propagation of embryonic stem cells
US5703055A (en) 1989-03-21 1997-12-30 Wisconsin Alumni Research Foundation Generation of antibodies through lipid mediated DNA delivery
US5143854A (en) 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof
US6040138A (en) 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays
US5800992A (en) 1989-06-07 1998-09-01 Fodor; Stephen P.A. Method of detecting nucleic acids
US5547839A (en) 1989-06-07 1996-08-20 Affymax Technologies N.V. Sequencing of surface immobilized polymers utilizing microflourescence detection
EP0430881A3 (en) 1989-11-29 1991-10-23 Ciba-Geigy Ag Photochromic compounds, process for their preparation and their use
US5288644A (en) 1990-04-04 1994-02-22 The Rockefeller University Instrument and method for the sequencing of genome
US5384253A (en) 1990-12-28 1995-01-24 Dekalb Genetics Corporation Genetic transformation of maize cells by electroporation of cells pretreated with pectin degrading enzymes
WO1993004169A1 (en) 1991-08-20 1993-03-04 Genpharm International, Inc. Gene targeting in animal cells using isogenic dna constructs
US5324633A (en) 1991-11-22 1994-06-28 Affymax Technologies N.V. Method and apparatus for measuring binding affinity
DE69322266T2 (de) 1992-04-03 1999-06-02 Perkin Elmer Corp Proben zusammensetzung und verfahren
US5340740A (en) 1992-05-15 1994-08-23 North Carolina State University Method of producing an avian embryonic stem cell culture and the avian embryonic stem cell culture produced by the process
US5702932A (en) 1992-07-20 1997-12-30 University Of Florida Microinjection methods to transform arthropods with exogenous DNA
US5589376A (en) 1992-07-27 1996-12-31 California Institute Of Technology Mammalian neural crest stem cells
US5453357A (en) 1992-10-08 1995-09-26 Vanderbilt University Pluripotential embryonic stem cells and methods of making same
US5503980A (en) 1992-11-06 1996-04-02 Trustees Of Boston University Positional sequencing by hybridization
US5523226A (en) 1993-05-14 1996-06-04 Biotechnology Research And Development Corp. Transgenic swine compositions and methods
US5858659A (en) 1995-11-29 1999-01-12 Affymetrix, Inc. Polymorphism detection
US5470710A (en) 1993-10-22 1995-11-28 University Of Utah Automated hybridization/imaging device for fluorescent multiplex DNA sequencing
GB9401833D0 (en) 1994-02-01 1994-03-30 Isis Innovation Method for discovering ligands
US5656610A (en) 1994-06-21 1997-08-12 University Of Southern California Producing a protein in a mammal by injection of a DNA-sequence into the tongue
US5736524A (en) 1994-11-14 1998-04-07 Merck & Co.,. Inc. Polynucleotide tuberculosis vaccine
US5843780A (en) 1995-01-20 1998-12-01 Wisconsin Alumni Research Foundation Primate embryonic stem cells
GB9507238D0 (en) 1995-04-07 1995-05-31 Isis Innovation Detecting dna sequence variations
US5661028A (en) 1995-09-29 1997-08-26 Lockheed Martin Energy Systems, Inc. Large scale DNA microsequencing device
US6013516A (en) 1995-10-06 2000-01-11 The Salk Institute For Biological Studies Vector and method of use for nucleic acid delivery to non-dividing cells
US5780448A (en) 1995-11-07 1998-07-14 Ottawa Civic Hospital Loeb Research DNA-based vaccination of fish
AU2253397A (en) 1996-01-23 1997-08-20 Affymetrix, Inc. Nucleic acid analysis techniques
US5945100A (en) 1996-07-31 1999-08-31 Fbp Corporation Tumor delivery vehicles
US5981274A (en) 1996-09-18 1999-11-09 Tyrrell; D. Lorne J. Recombinant hepatitis virus vectors
US6090622A (en) 1997-03-31 2000-07-18 The Johns Hopkins School Of Medicine Human embryonic pluripotent germ cells
US5994624A (en) 1997-10-20 1999-11-30 Cotton Incorporated In planta method for the production of transgenic plants
WO1999027076A1 (en) 1997-11-25 1999-06-03 Arc Genomic Research Pluripotent embryonic stem cells and methods of obtaining them
US5994136A (en) 1997-12-12 1999-11-30 Cell Genesys, Inc. Method and means for producing high titer, safe, recombinant lentivirus vectors
EP1129176A4 (de) 1998-11-09 2002-10-30 Es Cell Int Pte Ltd Embryonische stammzellen
US6645761B1 (en) 2000-12-22 2003-11-11 Stratagene Humanized polynucleotide sequence encoding Renilla mulleri green fluorescent protein
WO2007117573A2 (en) * 2006-04-07 2007-10-18 Cold Spring Harbor Laboratory Transgenic mice for identifying and assessing neural stem/ progenitor cells
WO2011026222A1 (en) * 2009-09-01 2011-03-10 Mcmaster University Transformed human pluripotent stem cells and associated methods
JP2011087475A (ja) * 2009-10-20 2011-05-06 Osaka Univ 未分化細胞の識別方法
WO2012035120A2 (en) * 2010-09-15 2012-03-22 Universiteit Leiden Screening method
TW201300783A (zh) * 2011-06-29 2013-01-01 Univ Nat Central 結合Nanog、Sox2、Lin28、Oct4啟動子及冷光基因之篩檢裝置及其方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011029798A1 (en) * 2009-09-09 2011-03-17 General Electric Company Composition and method for imaging stem cells

Also Published As

Publication number Publication date
US20180363070A9 (en) 2018-12-20
US20200347463A1 (en) 2020-11-05
US20170298453A1 (en) 2017-10-19
EP3194623A4 (de) 2018-03-14
EP3194623A1 (de) 2017-07-26
WO2016025510A1 (en) 2016-02-18

Similar Documents

Publication Publication Date Title
US20200347463A1 (en) Systems and methods to detect stem cell stress and uses thereof
Carbognin et al. Stat3 promotes mitochondrial transcription and oxidative respiration during maintenance and induction of naive pluripotency
Liu et al. RNA‐binding protein IGF2BP2/IMP2 is a critical maternal activator in early zygotic genome activation
Zhang et al. FOXO1 is an essential regulator of pluripotency in human embryonic stem cells
Puscheck et al. Molecular biology of the stress response in the early embryo and its stem cells
Kim et al. RETRACTED: Contribution of the PI3K/Akt/PKB signal pathway to maintenance of self-renewal in human embryonic stem cells
Andäng et al. Histone H2AX-dependent GABAA receptor regulation of stem cell proliferation
Toussaint et al. Stress‐induced premature senescence or stress‐induced senescence‐like phenotype: one in vivo reality, two possible definitions?
Oron et al. Cell fate regulation in early mammalian development
Xie et al. Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency
Sozen et al. The p38 MAPK signalling pathway is required for glucose metabolism, lineage specification and embryo survival during mouse preimplantation development
Schiffmacher et al. CDX2 regulates multiple trophoblast genes in bovine trophectoderm CT‐1 cells
Özbek et al. Sperm functional genome associated with bull fertility
Perrault et al. Global gene expression response to telomerase in bovine adrenocortical cells
Watkins et al. The preimplantation embryo: handle with care
Szczepny et al. Identification of Hedgehog signaling outcomes in mouse testis development using a hanging drop-culture system
Slater et al. Stress-induced enzyme activation primes murine embryonic stem cells to differentiate toward the first extraembryonic lineage
Clark et al. Primate primordial germ cells acquire transplantation potential by carnegie stage 23
Calder et al. Treatment with AICAR inhibits blastocyst development, trophectoderm differentiation and tight junction formation and function in mice
Van der Weijden et al. Molecular regulation of paused pluripotency in early mammalian embryos and stem cells
Schmidt et al. Placenta-derived macaque trophoblast stem cells: differentiation to syncytiotrophoblasts and extravillous trophoblasts reveals phenotypic reprogramming
Shi et al. Effect of FH 535 on in vitro maturation of porcine oocytes by inhibiting WNT signaling pathway
Lindgren et al. Loss of Pten causes tumor initiation following differentiation of murine pluripotent stem cells due to failed repression of Nanog
Li et al. Stress forces first lineage differentiation of mouse embryonic stem cells; validation of a high-throughput screen for toxicant stress
De Munck et al. mtDNA dynamics between cleavage-stage embryos and blastocysts

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20170224

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20180212

RIC1 Information provided on ipc code assigned before grant

Ipc: C12Q 1/68 20180101AFI20180206BHEP

Ipc: C12Q 1/6897 20180101ALI20180206BHEP

Ipc: C12N 5/00 20060101ALI20180206BHEP

Ipc: C12N 5/0735 20100101ALI20180206BHEP

Ipc: C12N 5/073 20100101ALI20180206BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20190218

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Ref document number: 602015076291

Country of ref document: DE

Free format text: PREVIOUS MAIN CLASS: C12Q0001680000

Ipc: G01N0033500000

GRAJ Information related to disapproval of communication of intention to grant by the applicant or resumption of examination proceedings by the epo deleted

Free format text: ORIGINAL CODE: EPIDOSDIGR1

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RIC1 Information provided on ipc code assigned before grant

Ipc: G01N 33/50 20060101AFI20210629BHEP

Ipc: C12N 5/073 20100101ALI20210629BHEP

Ipc: C12N 5/0735 20100101ALI20210629BHEP

Ipc: C12Q 1/6897 20180101ALI20210629BHEP

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20210812

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1461027

Country of ref document: AT

Kind code of ref document: T

Effective date: 20220115

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602015076291

Country of ref document: DE

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG9D

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20220105

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1461027

Country of ref document: AT

Kind code of ref document: T

Effective date: 20220105

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220505

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220405

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220405

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220406

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220505

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602015076291

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

26N No opposition filed

Effective date: 20221006

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20220811

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20220831

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20220831

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20220831

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20220811

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20220831

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20230828

Year of fee payment: 9

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20230825

Year of fee payment: 9

Ref country code: DE

Payment date: 20230829

Year of fee payment: 9

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20150811

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20220105